Novel differentiation-inhibitor

ABSTRACT

Amino acids constituting a physiologically active molecule human delta 2; a gene arrangement thereof; and an antibody thereto. The human delta 2 molecule, at least the amino acid sequence described in SEQ ID NO: 1 of the sequence listing, functions as a chemical useful for proliferation of undifferentiated blood cells and inhibition of differentiation, and hence can be utilized as pharmaceuticals and medical supplies.

[0001] This invention relates to a novel bioactive substance.

BACKGROUND OF THE INVENTION

[0002] Human blood and lymph contain various types of cells and eachcell plays important roles. For example, the erythrocyte carries oxygen;platelets have hemostatic action; and lymphocytes prevent frominfection. These various cells originate from hematopoietic stem cellsin the bone marrow. Recently, it has been clarified that thehematopoietic stem cells are differentiated to various blood cells,osteoclasts and mast cells by stimulation of various cytokines in vivoand environmental factors. In the cytokines, there have been found, forexample, erythropoietin (EPO) for differentiation to erythrocytes;granulocyte colony-stimulating factor (G-CSF) for differentiation toleukocytes; and platelet growth factor (mpl ligand) for differentiationto megakaryocytes which is a platelet producing cells, and the formertwo have already been clinically applied.

[0003] The undifferentiated blood cells are generally classified intotwo groups consisting of blood precursor cells which are destined todifferentiate to specific blood series and hematopoietic stem cellswhich have differentiation ability to all series and self-replicationactivity. The blood precursor cells can be identified by various colonyassays, however identification method for the hematopoietic stem cellshas not been established. In these cells, stem cell factor (SCF),interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin-6 (IL-6), interleukin-l (IL-1), granulocyte colonystimulating factor (G-CSF) and oncostatin M have been reported tostimulate cell differentiation and proliferation.

[0004] Trials for expansion of hematopoietic stem cells in vitro havebeen examined in order to replace bone marrow transplantation forapplying hematopoietic stem cell transplantation therapy or genetherapy. However, when the hematopoietic stem cells are cultured in thepresence of the above mentioned cytokines, multi-differentiationactivities and self-replication activities, which are originally in theposition of the hematopoietic stem cells, gradually disappeared and arechanged to the blood cell precursors which are only to differentiate tospecific series after 5 weeks of cultivation, and multi-differentiationactivity which is one of the specific features of the hematopoietic stemcells, is lost (Wagner et al. Blood 86, 512-523, 1995).

[0005] For proliferation of the blood precursor cells, single cytokineis not sufficient to effect, but synergistic action of several cytokinesare important. Consequently, in order to proliferate the hematopoieticstem cells in maintaining with specific features of the hematopoieticstem cells, it is necessary to add cytokines which suppressdifferentiation together with the cytokines which proliferate anddifferentiate the undifferentiated blood cells. In general, manycytokines, which stimulate proliferation or differentiation of cells,are known, but small numbers of cytokines, which suppressed celldifferentiation, are known. For example, leukemia inhibitory factor(LIF) has an action of proliferation of mouse embryonic stem cellswithout differentiation, but it has no action against the hematopoieticstem cells or blood precursor cells. Transforming growth factor (TGF-β)has suppressive action for proliferation against various cells, but nofixed actions against the hematopoietic stem cells or blood precursorcells.

[0006] Not only blood cells but also undifferentiated cells, especiallystem cells are thought to be involved in tissue regeneration. Theseregeneration of tissues and proliferation of undifferentiated cells ineach tissue can be applied in various ways by referring to the knownreference (Katsutoshi Yoshizato, Regeneration—a mechanism ofregeneration, 1996, Yodosha Publ. Co.).

[0007] Notch is a receptor type membrane protein, which involves inregulation of nerve cells differentiation found in Drosophila.Homologues of the Notch are found in various animal kinds exceeding tothe invertebrate and vertebrate including nematode (Lin-12). Xenopuslaevis (Xotch), mouse (Motch) or human (TAN-1).

[0008] Ligand of the Notch in Drosophila is known. These are DrosophilaDelta (Delta) and Drosophila Serrate (Serrate). Notch ligand homologuesare found in various animal kinds as similar to the Notch of receptors(Artavanis-Tsakonas et al., Science 268, 225-232, 1995).

[0009] Human Notch homologue, TAN-1 is found widely in the tissues invivo (Ellisen et al., Cell 66, 649-661, 1991). Three Notch analogousmolecules other than TAN-1 are reported (Artavanis-Tsakonas et al.,Science 268, 225-232, 1995). Expression of TAN-1 was also observed inCD34 positive cells in blood cells by PCR (Polymerase Chain Reaction)(Milner et al., Blood 83, 2057-2062, 1994). However, in relation tohumans, gene and amino acid sequences of human Delta and human Serrate,which are thought to be the Notch ligand, have not been reported asscientific reports in April 1997.

[0010] In Drosophila Notch, binding with the ligand was studied andinvestigated in details, and it was found that the Notch can be bound tothe ligand with Ca++ at the binding region, which is a repeated aminoacid sequence No. 11 and No. 12 in the amino acid sequence repeat ofEpidermal Growth Factor (EGF) like repeating (Fehon et al., Cell 61.523-534, 1990, Rebay et al., ibid. 67, 687-699, 1991 and InternationalPublication WO 92/19734). EGF-like repeated sequences are conserved inNotch homologues of the other species. Consequently, the same mechanismin binding with ligand is estimated. An amino acid sequence which iscalled as DSL (Delta-Serrate-Lag-2) near the amino acid terminal, andEGF-like repeated sequence as like in the receptor are conserved in theligand (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).

[0011] EGF-like sequence has been found in thrombomodulin (Jackman etal., Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986), low densitylipoprotein (LDL) receptor (Russell et al., Cell 37, 577-585, 1984), andblood coagulating factor (Furie et al., Cell 53, 505-518, 1988), and isthought to play important roles in extracellular coagulation andadhesion.

[0012] Recently, the vertebrate homologues of the cloned DrosophilaDelta were found in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1),and it has reported that X-Delta-1 had acted through Xotch in thegeneration of the protoneuron (Henrique et al., Nature 375, 787-790,1995 and Chitnis et al., ibid. 375, 761-766,1995). Vertebrate homologueof Drosophila Serrate was found in rat as rat Jagged (Jagged) (Lindsellet al., Cell 80, 909-917, 1995). According to the Lindsell et al., mRNAof the rat Jagged is detected in the spinal cord of fetal rats. As aresult of cocultivation of a myoblast cell line that is forced excessexpressed rat Notch with a rat Jagged expression cell line, suppressionof differentiation of the myoblast cell line is found. However, the ratJagged has no action against the myoblast cell line without forcedexpression of the rat Notch.

[0013] A hypothesis has been set up so that Notch and its ligand have anaction of differential regulation not only for neuroblasts andmyoblasts, but also for various undifferentiated cells, especially bloodundifferentiated cells. However, as far as clinical applications inhumans, prior known different species such as chicken or Xenopus laevostype Notch ligand have problems with species specificities andantigenicities. Consequently, obtaining prior unknown human Notch ligandis essentially required. The inventor suspected that a molecule havingDSL domain and EGF-like domain which are common to Notch ligandmolecules and a ligand of the human Notch (TAN-1 etc.), which is a humanDelta homologue (hereinafter designates as human Delta) and humanSerrate homologue (hereinafter designates as human Serrate), may befound. In addition, these findings may be a candidate for a drug usefulfor differential regulation of undifferentiated cells.

[0014] As a result, in the previous patent application, a gene cloningof three types of molecules including human Delta-1, human Serrate-1 andhuman Serrate-2 molecules as the human Notch ligand molecules was made,and it was found that these molecules have an action on bloodundifferentiated cells. (Refer to WO 97/19172Differentiation-suppressive polypeptide and WO 98/02458Differentiation-inhibitor). As for the human Notch ligand molecule,according to the recent report, partial gene and partial amino acidsequences of the human Delta-1 like molecule, which are, however,incomplete with respect to the specification and disclosure of thefull-length sequence, have been disclosed in the InternationalPublication WO 97/01571. Further, WO 96/27610 discloses total lengthgene and total length amino acid sequences of human Serrate-1(humanJagged-1). Also, WO 96/27610 discloses partial length gene andpartial length amino acid sequences of human Serate-2 (human Jagged-2).This gene sequence might have erroneous sequences and this gene sequencegenerates frame shift, which results completely different amino acidsequence of our WO 98/02458, Differentiation-inhibitor. In addition, thesaid prior arts did not disclose gene cloning of amino terminals.Consequently, the gene sequences and amino acid sequences areincomplete. As a result of searching the gene sequence database,Genebank Release 98 (December 1996), there are four entries about humanSerrate-1, i.e. Registered No. HSU61276, HSU3936, HSU77720 and HSU77914,however no other human Notch ligand molecules are found in the saiddatabase.

[0015] The present invention elucidates the gene sequence and amino acidsequence of novel Notch ligand molecules. Novel Notch ligand moleculesand novel therapeutic uses for these molecules are also provided.

[0016] In order to search novel human Notch ligands, cross hybridizationusing the human Delta-1 gene was performed.

[0017] To obtain the human Delta-1 gene, methods used in the referentialexamples 1 and 2, and WO 97/19172 can be applied. Transformed cells, inwhich a vector pUCDL-1 containing cDNA coding total amino acid sequenceof human Delta-1, i.e. DNA containing sequence from No. 179 to No. 2347in SEQ ID NO: 8, is inserted into E. coli JM109, have been deposited inthe National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, in Higashi 1-1-3, Tsukuba, Ibaragi-ken, Japan as permanentculture collection E. coli: JM109-pUCDL-1F. Date of deposition was Oct.28, 1996 as deposition No. FERM BP-5728.

[0018] Various lengths of partial genes of this human Delta-1 gene wereprepared. Using these partial lengths of genes as probes, numerous cDNAlibraries were screened under various hybridization conditions todetermine novel Notch ligands by cross hybridization methods.

[0019] As a result of extensive studies, the isolation of cDNA codingamino acid sequences of novel human Delta-2 have been achieved, a novelmolecule having DSL domain common to Notch ligand molecules from humanfetal lung cDNA library, and have prepared the expression systems ofprotein having various forms using the cDNA. Also we have establishedpurification methods of the proteins which were purified and isolated.

[0020] Amino acid sequences of novel human Delta-2 are shown in thesequence listings, SEQ ID NO: 1-3. DNA sequence coding these sequencesis shown in the sequence listing, SEQ ID NO: 4.

[0021] Physiological actions of the these prepared proteins weresearched by using many types of cells, for example nerveundifferentiated cells, preadipocytes, hepatocytes, myoblasts, skinundifferentiated cells, blood undifferentiated cells and immuneundifferentiated cells. As a result, it has been found that novel humanDelta-2 had a differentiation-suppressive action againstundifferentiated blood cells, and had a physiological action to maintainan undifferentiated state. Further, it has been found that the moleculehas growth suppressive action against vascular endothelial cells.

[0022] No significant toxic actions were noted in the toxicity studieson mice, and useful pharmaceutical effects were suggested. Consequently,the pharmaceutical preparations containing the molecule of the presentinvention, medium containing the molecule of the present invention, andthe device immobilized with the molecule of the present invention arenovel drugs and medical materials which can maintain the bloodundifferentiated cells in the undifferentiated condition. Antibodyagainst human Delta-2 is prepared by using antigen of the said humanDelta-2, and purification method of the said antibodies is established.The present invention has completed accordingly.

[0023] The present invention relates to a polypeptide comprising atleast amino acid sequence of SEQ ID NO: 1 of the sequence listing, apolypeptide comprising at least amino acid sequence of SEQ ID NO: 2 ofthe sequence listing, and a polypeptide comprising at least amino acidsequence of SEQ ID NO: 3 of the sequence listing. The present inventionalso relates to the said polypeptides having differentiation suppressiveaction against undifferentiated cells, the said polypeptides in whichthe undifferentiated cells are undifferentiated cells except for thoseof brain and nervous system or muscular system, the said polypeptides inwhich the undifferentiated cells are undifferentiated blood cells, andthe said polypeptides acting on vascular cells. The present inventionalso relates to a pharmaceutical composition comprising the saidpolypeptides, and the said pharmaceutical composition havingdifferentiation suppressive action against cells, the saidpharmaceutical composition in which the cells are undifferentiated bloodcells, and the said pharmaceutical composition having regulatory actionagainst vascular cells. The present invention further relates to a cellculture medium comprising the said polypeptides, the cell culture mediumin which the cell is undifferentiated blood cell, and a material havingimmobilized thereto the polypeptide. Further, the present inventionrelates to a method for culturing cells using the cell culture medium orthe material, and the method in which the cells are undifferentiatedblood cells.

[0024] The present invention further relates to a DNA coding at least anamino acid sequence of the sequence listing, SEQ ID NO: 1, said DNAcoding at least an amino acid sequence of the sequence listing, SEQ IDNO: 2, the DNA coding at least an amino acid sequence of the sequencelisting, SEQ ID NO: 3, the DNA having a base sequence from 355 to 927 ofthe sequence listing, SEQ ID NO: 4, the DNA having base sequence from355 to 1854 of the sequence listing, SEQ ID NO: 4 and the DNA havingbase sequence from 355 to 2331 of the sequence listing SEQ ID NO: 4. Thepresent invention still further relates to a recombinant DNA comprisinga DNA selected from the group consisting of the DNAs having ligated to avector DNA which can express said DNA in the host cell, a celltransformed by the recombinant DNA, a method for culturing human cellswith the said cells, and a process for production of said polypeptide byculturing the said cells and isolating the compound produced in thecultured mass. The present invention still more further relates to anantibody specifically recognizing a polypeptide having an amino acidsequence of the sequence listing, SEQ ID NO: 3.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Preparation of cDNA necessary for gene manipulation, expressionanalysis by Northern blotting, screening by hybridization, preparationof recombinant DNA, determination of DNA base sequence and preparationof cDNA library, all of which are series of molecular biologicalexperiments, can be performed according to a description of theconventional textbook for the experiments. The above conventionaltextbook of the experiments is, for example, Maniatis et al. ed.Molecular Cloning, A laboratory manual, 1989, Eds., Sambrook, J.,Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press.

[0026] A polypeptide of the present invention has at least polypeptidesin the sequence listing SEQ ID NO: 1-3. A mutant and allele, whichnaturally occur in the nature, are included in the polypeptide of thepresent invention unless the polypeptides of the sequence listing, SEQID NO: 1-3 loses their properties. Modification and substitution ofamino acids are described in details in the patent application by thename of Benntt et al. (National Unexam. Publ. WO 96/2645) and can beprepared according to the description thereof.

[0027] A DNA sequence coding polypeptides of the sequence listing, SEQID NO: 1-3 is shown in the sequence listing, SEQ ID NO: 4, together withtheir amino acid sequences. In these DNA sequences, even if amino acidlevel mutation is not generated, naturally isolated chromosomal DNA orcDNA thereof may have a possibility to mutate in the DNA base sequenceas a result of degeneracy of genetic code without changing amino acidsequence coded by the DNA. A 5′-untranslated region and 3′-untranslatedregion do not involve in amino acid sequence determination of thepolypeptide, so DNA sequences of these regions are easily mutated. Thebase sequence obtained by these regeneracies of genetic codes isincluded in the DNA of the present invention.

[0028] Undifferentiated cells in the present invention are defined ascells, which can growth by specific stimulation, and cells, which can bedifferentiated to the cells having specific functions as a result of thespecific stimulation. These include undifferentiated cells of the skintissues, undifferentiated cells of the brain and nervous systems,undifferentiated cells of the muscular systems and undifferentiatedcells of the blood cells. These cells include the cell ofself-replication activity which is called as stem cells, and the cellhaving an ability to generate the cells of these lines. Thedifferentiation-suppressive action means suppressive action forautonomous or heteronomous differentiation of the undifferentiatedcells, and is an action for maintaining undifferentiated condition. Thebrain and nervous undifferentiated cells can be defined as cells havingability to differentiate to the cells of the brain or nerve havingspecific functions by specific stimulation. The undifferentiated cellsof the muscular systems can be defined as cells having ability todifferentiate to the muscular cells having specific functions byspecific stimulation. The blood undifferentiated cells in the presentinvention can be defined as cell groups consisting of the bloodprecursor cells which are differentiated to the specific blood seriesidentified by blood colony assay, and hematopoietic stem cells havingdifferentiation to every series and self-replication activities.Further, in the present invention, vascular cells is defined as generalnomination for cells constituting blood vessels, in which vascularendothelial cells is major constituting cells.

[0029] The amino acid sequence in the sequence listing, SEQ ID NO: 1 isa sequence of the active center of the novel human Delta-2 of thepresent invention, from which the signal peptide is deleted, i.e. aminoacid sequence from the amino terminal to DSL domain, and corresponds toan amino acid No. 1 to 191 in SEQ ID NO: 3 of the matured full lengthaminoacid sequence of the novel human Delta-2 of the present invention.

[0030] The amino acid sequence in SEQ ID NO: 2 is amino acid sequence ofextracellular domain of the novel human Delta-2 of the presentinvention, from which the signal peptide is deleted, and corresponds toan amino acid No. 1 to 500 in SEQ ID NO: 3 of the matured full lengthamino acid sequence of the novel human Delta-2 of the present invention.

[0031] The amino acid sequence of SEQ ID NO: 3 is the matured fulllength amino acid sequence of the novel human Delta-2 of the presentinvention.

[0032] The sequence of SEQ ID NO: 4 is cDNA sequence and total aminoacid sequence of the novel human Delta-2 of the present invention, whichcorresponds to the coding region of the said cDNA.

[0033] The sequence of SEQ ID NO: 5 is DNA sequence which codes FLAGpeptide and amino acid sequence of FLAG peptide used in the presentinvention.

[0034] The sequences of SEQ ID NOs: 6 and 7 are DNA sequences of primersused in referential example 1.

[0035] The sequence of SEQ ID NO: 8 is the cDNA sequence and total aminoacid sequence of human Delta-1 used in the present invention.

[0036] The sequences of SEQ ID NOs: 9, 10, 12 and 13 are DNA sequencesof primers used in example 1.

[0037] The sequence of SEQ ID NO: 11 is a DNA sequence of a probe usedin example 1.

[0038] The sequence of SEQ ID NO: 14 is a DNA sequence of a probe usedin examples 1 and 2.

[0039] The sequences of SEQ ID NOs: 15 to NO: 24 are DNA sequences ofprimers used in example 3.

[0040] The left and right ends of the amino acid sequences in thesequence listings indicate amino terminal (hereinafter designates asN-terminal) and carboxyl terminal (hereinafter designates asC-terminal), respectively, and the left and right ends of the nucleotidesequences are 5′-terminal and 3′-terminal, respectively.

[0041] Cloning of unknown human Notch ligand gene can be performed bythe following method. During the evolution of the organisms, a part ofamino acids sequences and gene sequences of the human Notch ligand isconserved. Cloning can be theoretically possible by using the otherNotch legand molecule as a probe. However, in such the crosshybridization, there are many problems, for example, what part ispreferable for the probe or how to set up condition for hybridization,and are not so simple. Further, since the cross hybridization processtends to make cloning many numbers of similar genes simultaneously, ittakes much times for gene sequence analysis, consequently identificationof the objective molecules from the cloned genes is quite difficult.

[0042] More than 10 gene fragments have been prepared from the humanDelta-1 gene. By using said probes, screenings of cDNA libraries whichoriginate from more than ten different organs were performed undernumerous hybridization conditions and washing conditions. A novel Deltalike molecule has been sought after.

[0043] In the plaque hybridization, clones can be obtained by isotopelabeling and non-isotope labeling with the probe. Isotope labeling canbe performed by, for example, terminal labeling by using [32P]γ-ATP andT4 polynucleotide kinase, or other labeling methods such as nicktranslation or primer extension method can be applied.

[0044] As a result, in example 1, a screening of a human fetal lung cDNAlibrary was prepared using a partial gene of the full length gene of thehuman Delta-1 as shown in SEQ ID NO: 8 in the sequence listing, i.e.gene sequence shown in SEQ ID NO: 11 in the sequence listing, as aprobe. As a result of the first screening, about 120 positive plaqueswere isolated, and in the second screening, about 80 positive plaqueswere cloned, then gene sequences of these clones were determined. Mostof these cloned genes were the human Delta-1 gene used as a probe. Amongthem, five clones were found as the novel human Delta-2 gene, which issimilar to the human Delta-1 gene, and the objective novel Notch ligandmolecule was found. these cloned genes were the human Delta-1 gene usedas a probe. Among them, five clones were found as the novel humanDelta-2 gene, which is similar to the human Delta-1 gene, and objectivenovel Notch ligand molecule was found.

[0045] Among the above five clones, since there were no signal sequenceand no amino terminal sequence, a new probe having a gene sequence asshown in SEQ ID NO: 14 was prepared in order to obtain the full lengthgene. Further, a screening of said human fetal lung cDNA library wasrepeated with this probe. As a result, cloning of the cDNA coding forthe full length of gene was accomplished.

[0046] This sequence was compared with the databse (Genbank Release 89,December 1996), and found that these were novel sequence.

[0047] Examples of plasmids integrated with cDNA are, for (example,other than pBluescript KS described in example 1, E. coli originatedpBR322, pUC18, pUC19, pUC118 and pUC119 (Takara Shuzo Co. Japan), butthe other plasmids can be used, if they can replicate and proliferate inthe host cells. Examples of phage vectors integrated with cDNA are, forexample, λgt10 and λgt11, but the other vectors can be used, if they cangrowth in the host cells. The thus obtained plasmids are transduced intosuitable host cells such as genus Escherichia and genus Bacillus usingcalcium chloride method. Examples of the above genus Escherichia areEscherichia coli K12HB101, MC1061, LE392 and JM109. Example of the abovegenus Bacillus is Bacillus subtilis MI114. Phage vector can beintroduced into the proliferated E. coli by the in vitro packagingmethod (Enquist and Sternberg, Meth. Enzymol., 68, 281-, 1979).

[0048] The said amino acid sequence was analyzed hydrophobic part andhydrophilic part from amino acid sequence according to the method ofKyte-Doolittle (J. Mol. Biol. 151: 105, 1982). As a result, the novel

[0049] human Delta-2 of the present invention is expressed on cells ascell membrane protein having one transmembrane domain.

[0050] According to an analysis of the amino acid sequence of the novelhuman Delta-2, an amino acid sequence of a precursor of the novel humanDelta-2 consists of a 685 amino acid residue shown in the sequencelisting, SEQ ID NO: 4, and the signal peptide domain is estimated tocorrespond to the amino acid sequence of 26 amino acids residue from No.−26 methionine to No. −1 glycine of the sequence listing; extracellulardomain: 500 amino acids residue from No. 1 serine to No. 500 serine;transmembrane domain: 26 amino acids residue from No. 501 phenylalanineto No. 526 valine; and intracellular domain: 133 amino acids residuefrom No. 527 arginine to No. 659 valine. The domain construction isestimated from the amino acid sequences, and an actual presence forms onthe cells. Furthermore, a solution may be possible that differs from theabove structure, and structural amino acid sequence of each domainhereinabove as defined by possibly changing 5 to 10 amino acids of thesequence.

[0051] N-terminal amino acid sequence of the human Delta-2 polypeptide,which is expressed in COS-7 cells, produced and purified as described inexample 5, has at least the amino acid sequence started from No, 1serine in the sequence listing, SEQ ID NO: 2. Similarly, identicalN-terminal can be expected, if the said peptide is expressed in theother animal cells.

[0052] According to a comparison in the full length amino acid sequenceof the novel human Delta-2 of the present invention with other Notchligand molecules, which has been reported by April 1997, the homologywith human Delta-1 (amino acid sequence of SEQ ID NO: 8 in the sequencelisting) as a molecule originated from human is 48.5%; with Serrate-1(Genbank HSU61276 and HSU73939) is 40.3%; and with human Serrate-2(Japanese Patent Appln. No. 8-18622, Differentiation-suppressivepolypeptide in the name of the present inventors) is 42,7%. Thehomologies with Delta of other vertebrates are: mouse Delta-1 (D111,Genbank MMDELTA1) 48.7%: flog Delta-1 (Genbank XELXDEL) 47.0%; flogDelta-2 (Genbank XLU70843) 49.7%, and chicken Delta-1 (Genbank GGU26590)47.9%

[0053] As the result, the human Delta-2 of the present invention is anovel molecule, which has never been reported in humans or in otherbiological homologues, and is a novel substance having an amino acidsequence different from these substances, and is a novel substance whichhas been elucidated for the first time by the present inventor.Moreover, no polypeptide having the same amino acid sequence as thenovel human Delta-2 has been found by a homology search in otherorganisms.

[0054] The homologues of Notch ligand have an evolutionary conservedcommon sequence, i.e. a DSL sequence and repeated EGF-like sequence. Asa result of a comparison with the novel human Delta-2 and human Delta-1,these conserved amino acid sequences of the novel human Delta-2 areestimated.

[0055] Namely, DSL sequence corresponds to 43 amino acids residue fromNo. 149 cystine to No. 191 cystine of the amino acid sequence in thesequence listing, SEQ ID NO. 4. EGF-like sequence exists with 8 repeatswherein, in the amino acid sequence in the sequence listing, SEQ ID NO:4, the first EGF-like sequence corresponds to the sequence from No. 196cystine to No. 224 cystine: the second EGF-like sequence corresponds tothe sequence from No. 227 cystine to No. 225 cystine; the third EGF-likesequence corresponds to the sequence from No. 262 to No. 295 cystine:the fourth EGF-like sequence corresponds to the sequence from No. 302cysteine to No. 333 cysteine; the fifth EGF-like sequence corresponds tothe sequence from No. 340 cysteine to No. 373 cysteine; the sixthEGF-like sequence corresponds to the sequence from No. 380 cysteine toNo. 411 cysteine; the seventh EGF-like sequence corresponds to thesequence from No. 418 cysteine to No. 449 cysteine; and the eighthEGF-like sequence corresponds to the sequence from No. 458 cysteine toNo. 491 cysteine.

[0056] A part of sugar chain attached is estimated from amino acidsequence of the novel human Delta-2 may be No. 82, 157, 179 and 367asparagine residue in the sequence listing, SEQ ID NO: 4 as a possiblebinding site of N-glycoside bonding for N-acetyl-D-glucosamine.O-glycoside bond of N-acetyl-D-galactosamineis estimated to be a serineor threonine residue rich part. Protein bound with sugar chain isgenerally thought to be stable in vivo and to have strong physiologicalactivity. Consequently, in the amino acid sequence of polypeptide havingsequence of the sequence listing SEQ ID NO: 1, 2 or 3, polypeptideshaving N-glucoside or O-glucoside bond with sugar chain ofN-acetyl-D-glucosamine or N-acetyl-D-galactosamine is included in thepresent invention. As shown in example 5, if the human Delta-2 of thepresent invention is expressed by gene inserted COS-7 cell, at leastmore than two forms are expressed due to attached sugar chain as theproteins having different molecular weight.

[0057] As a result of studies on binding of Drosophila Notch and itsligand, amino acid region necessary for binding with ligand ofDrosophila Notch with the Notch is from N-terminal to DSL sequence ofthe matured protein, in which signal peptide is removed (InternationalPublication WO 92/19734). Further, similarly, studies using Nematode byFitzgerald and Greenwald (Development, 121, 4275-4282, 1995) clearlyindicate that Notch ligand like molecule APX-1 required for Notch likereceptor activation is sufficient from amino terminal to DSL domain inthe full length sequence.

[0058] This fact indicates that a domain necessary for expression ofligand action of human Notch ligand molecule is at least the DSL domain,i.e. a domain containing amino acid sequence from No. 149 cycteine toNo. 191 cycteine in the sequence listing, SEQ ID NO: 1, and a domain atleast necessary for expression of ligand action of human Delta-2 isnovel amino acid sequence shown in the sequence listing, SEQ ID NO: 1,further a domain at least necessary for expression of ligand action ofhuman Delta-2 is novel amino acid sequence shown in the sequencelisting, SEQ ID NO: 2.

[0059] As shown in example 2, mRNA of the human Delta-2 can be detectedby using DNA coding a part or full of gene sequence in the sequencelisting, SEQ ID NO: 4. For example, a method for detection of expressionof these genes can be achieved by applying with hybridization or PCR byusing complementary nucleic acids of above 12mer or above 16mer,preferably above 20mer having nucleic acid sequence of a part ofswquence in the sequence listing, SEQ ID NO: 4, i.e. antisense DNA orantisense RNA, its methylated, methylphosphated, deaminated orthiophosphated derivatives. Using the same method, detection ofhomologues of the gene of other organisms such as mice or gene cloningcan be achieved. Further cloning of genes in the genome including humanscan be made. Using these genes cloned by such like methods, furtherdetailed functions of the human Delta-2 can be clarified. For example,using the recent gene manipulation techniques, every methods includingtransgenic mouse, gene targeting mouse or double knockout mouse in whichgenes relating to the gene of the present invention are inactivated, canbe applied. If abnormalities in the genome of the present gene arefound, application to gene diagnosis and gene therapy can be made.

[0060] A transformant in which vector pBSDL-2, which contains cDNAcoding total amino acid sequence of the novel human Delta-2 of thepresent invention, is transformed into E. coli JM109, has been depositedin the National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, of 1-1-3, Higashi, Tsukuba-shi, Ibaragi-ken, Japan, as E.coli: JM109-pBSDL-2. Date of deposit was May 9, 1997, and deposition No.is FBRM BP-5941.

[0061] Process for production of the novel human Delta-2 polypeptide canbe performed, as shown in example 3, by using expression vector pcDNA 3.Production and purification of various forms of the novel human Delta-2polypeptide using cDNA, which codes amino acid sequence of the novelhuman Delta-2 isolated by the above method are known in the references(Kriegler, Gene Transfer and Expression-A Laboratory Manual, StocktonPress, 1990 and Yokota et al. Biomanual Series 4, Gene Transfer andExpression and Analysis, Yodosha Co., 1994). A cDNA coding the aminoacid sequence of the isolated said human Delta-2 is ligated topreferable expression vector and it is produced in the host cells ofeukaryotic cells such as animal cells and insect cells or prokaryoticcells such as bacteria.

[0062] In the expression of the novel human Delta-2 of the presentinvention, DNA coding polypeptide of the present invention may have thetranslation initiation codon in 5′-terminal and translation terminationcodon in 3′-terminal. These translation initiation codon and translationtermination codon can be added by using preferable synthetic DNAadapter. Further for expression of the said DNA, promoter is ligated inthe upstream of the DNA sequence. Examples of vector are plasmidoriginated from the above E. coli, plasmid originated from Bacillus,plasmid originated from yeast or bacteriophage such as λ-phage andanimal viruses such as retrovirus and vaccinia virus.

[0063] Examples of promoters used in the present invention are anypromoters preferable for corresponding to the host cells used in geneexpression.

[0064] In case that the host cell in the transformation is genusEscherichia, tac-promoter, trp-promoter and lac-promoter are preferable,and in case of host of genus Bacillus, SP01 promoter and SP02 promoterare preferable, and in case of host of yeast, PGK promoter, GAP promoterand ADH promoter are preferable.

[0065] In case that the host cell is animal cell, a promoter originatedfrom SV40, promoter of retrovirus, metallothionein promoter and heatshock promoter can be applied.

[0066] Expression of the polypeptide of the present invention can bemade by using only DNA coding the amino acid sequence of the sequencelisting, SEQ ID NO: 1, 2 or 3. However, the protein added with specificfunction can be produced by using DNA, to which added cDNA coding theknown antigen epitope for easier detection of the produced polypeptideor added cDNA coding the immunoglobulin Fc for forming multimer.

[0067] As shown in Example 3, we have prepared expression vectors, whichexpress extracellular proteins of the novel human Delta-2, as follow:

[0068] 1) DNA coding the amino acids from No. 1 to 500 in amino acidsequence in the sequence listing, SEQ ID NO: 2;

[0069] 2) DNA coding chimera protein, to which added polypeptide having8 amino acids, i.e. an amino acid sequence consisting of Asp Tyr Lys AspAsp Asp Asp Lys (hereinafter designates FLAG sequence, an example of DNAsequence coding the same is shown in the sequence listing, SEQ ID NO:5), in the C-terminal of the amino acids from No. 1 to 500 in amino acidsequence in the sequence listing, SEQ ID NO: 2; and

[0070] 3) DNA coding chimera protein, to which added Fc sequence fromthe hinge region of human IgG1 in the C-terminal of the amino acids fromNo. 1 to 500 in amino acid sequence in the sequence listing, SEQ ID NO:2, is ligated to each separately with the expression vector pcDNA 3(INVITROGEN Corp., U.S.A.), then the expression vector expressing theextracellular region of the novel human Delta-2 is prepared.

[0071] The expression vector for expression of full length of the novelhuman Delta-2 can be prepared as follows:

[0072] 4) DNA coding amino acids from No. 1 to 659 in the sequencelisting. SEQ ID NO: 3 and

[0073] 5) DNA coding chimera protein, to which added polypeptide havingFLAG sequence in the C-terminal of amino acids from No. 1 to 659 in thesequence listing, SEQ ID NO: 3 are ligated individually with theexpression vector pcDNA to prepare the expression vector, which canexpress full length of the novel human Delta-2.

[0074] The transformants are prepared by using these expression plasmidscontaining DNA coding the thus constructed said human Delta-2.

[0075] Examples of the host are genus Escherichia, genus Bacillus, yeastand animal cells. Examples of animal cells are simian cell COS-7 andVero, Chinese hamster cell CH0 and silk worm cell SF9.

[0076] As shown in Example 4, the expression vectors of the above 1) -5)are transduced individually; the novel human Delta-2 is expressed inCOS-7 cell (obtainable from the Institute of Physical and ChemicalResearch, Cell Development Bank, RCB0539), and the transformants, whichare transformed by these expression plasmids, can be obtained. Further,the novel human Delta-2 polypeptide can be produced by culturing thetransformants under preferable culture condition in medium by knownculture method.

[0077] As shown in Example 5, the novel human Delta-2 polypeptide can beisolated and purified from the above cultured mass, in general, by thefollowing methods.

[0078] For extraction of the substance from cultured microbial cells orcells, microbial cells or cells are collected by known methods such ascentrifugation after the cultivation, suspended in preferable buffersolution, disrupted the microbial cells or cells by means ofultrasonication, lysozyme and/or freeze-thawing and collected crudeextract by centrifugation or filtration. The buffer solution may containprotein-denaturing agents such as urea and guanidine hydrochloride orsurface active agents such as Triton-X. In case of secretion in thecultured solution, the cultured mass is separated by the known methodsuch as centrifugation to separate from microbial cells or cells and thesupernatant solution is collected.

[0079] The thus obtained novel human Delta-2, which is contained in thecell extracts or cell supernatants, can be purified by known proteinpurification methods. During the purification process, for confirmationof existence of the protein, in case of the fused proteins such as theabove FLAG and human IgGFc, they can be detected by immunoassay usingantibody against known antigen epitope and can be purified. In case ofnot to express as such the fused protein, the antibody in Example 6 canbe used for detection.

[0080] Antibodies, which specifically recognize human Delta-2, can beprepared as shown in Example 6. Antibodies can be prepared by themethods described in the reference (Antibodies a laboratory manual, E.Harlow et al., Cold Spring Harbor Laboratory) or recombinant antibodiesexpressed in cells by using immunoglobulin genes isolated by genecloning method. The thus prepared antibodies can be used forpurification of the novel human Delta-2. The human Delta-2 can bedetected and assayed by using antibodies which recognize specificallynovel human Delta-2 as shown in Example 6, and can be used fordiagnostic agents for diseases accompanied with abnormal differentiationof cells such as malignant tumors.

[0081] More useful purification method is the affinity chromatographyusing antibody. Antibodies used in this case are antibodies described inExample 6. For fused protein, antibodies against FLAG in the case ofFLAG, and protein G or protein A in the case of human IgGFc as shown inExample 5.

[0082] Physiological functions of the thus purified human Delta-2protein can be identified by various assay methods, for example,physiological activity assaying methods using cell lines and animalssuch as mice and rats, assay methods of intracellular signaltransduction based on molecular biological means, binding with Notchreceptor etc.

[0083] Actions for blood undifferentiated cells have been observed byusing IgG1 chimera protein of novel human Delta-2. As a result, it hasbeen found that, as shown in example 7, in undifferentiated umbilicalcord derived blood cells, in which the CD34 positive cell fraction isconcentrated, the novel human Delta-2 has suppressive action of colonyforming action against blood undifferentiated cells, which show colonyformation in the presence of cytokines.

[0084] Further as shown in example 8, it has been found that byevaluating LTC-IC (Long-term Culture-Initiating Cells), which arepositioned from most undifferentiated blood stem cells in the humanblood undifferentiated cells, after culturing the undifferentiatedumbilical cord derived blood cells, in which the CD34 positive cellfraction is concentrated, in the presence of the human Delta-2 withvarious cytokines in serum-free medium, the human Delta-2 has anactivity to maintain a number of LTC-IC. Further, example 9 shows thatthe human Delta-2 is Bound with human blood undifferentiated cells.

[0085] The results indicate that the human Delta-2 suppressesdifferentiation of blood undifferentiated cells, and this action isobviously effective for cells from blood stem cells to colony formingcells. These physiological actions are essential for in vitroproliferation of blood undifferentiated cells. Especially, cellscultured in the medium containing human Delta-2 are efficient inrecovery of suppression of bone marrow after administration of antitumoragents, accordingly in vitro expansion of hemopoietic stem cells may bepossible if other conditions would be completed. Further pharmaceuticalscontaining the polypeptide of the present invention have protective andreduced actions against the bone marrow suppressive action due toadverse effects of antitumor agents.

[0086] In these experiments, the LTC-IC maintaining activity and bindingaction for blood cells of the novel human Delta-2 of the presentinvention are stronger than those of the human Delta-1 (WO 97/19172),which has same action as shown by the present inventors.

[0087] As shown in example 9, IgG1 chimera protein of the human Delta-2is bound with CD34 positive blood undifferentiated cells. By thisbinding activity, the polypeptide of the present invention can be usedfor isolation and detection of cells. Although the isolation method canbe performed by a method using flow cytometer as described in example 9,a method using materials, to which polypeptide of the present inventionis immobilized, as described in example 11 may be more convenient.Consequently, cell isolation method using polypeptide of the presentinvention is included in the present invention. Further, cell isolationmethod using a material, to which polypeptide of the present inventionis immobilized, and device for cell isolation applied with the saidisolation method is also includedin the present invention. Any cellisolation method using antibodies described in the references isapplicable to these isolation devices and isolation methods. Forexample, Dynabeads of Dynal Corp., Norway, which is a method usingcombination of magnetic beads and antibodies, can be used.

[0088] Further, as shown in example 12, IgG1 chimera protein of thenovel human Delta-2 of the present invention has suppressive actionagainst proliferation of intravascular endothelial cells and hasinhibitory action against vascularization. Consequently, the polypeptideof the present invention can be used as therapeutic agents for diseasesand disease states, which may be cured by suppressing vascularization asproposed by Folkman and Klagsbrun (Science 235, 442-447, 1987). Concreteexamples of use are described in the above reference, and are, forexample, diseases including malignant tumors.

[0089] Suppressive action for differentiation of cells in theundifferentiated cells other than blood cells is expected andstimulating action for tissue regeneration can be expected.

[0090] In the pharmaceutical use, polypeptides of the present inventionare lyophilized with adding preferable stabilizing agents such as humanserum albumin, and are used in dissolved or suspended condition withdistilled water for injection when it is in use. For example,preparation for injection or infusion at the concentration of 0.1-1000μg/ml may be provided. A mixture of the compound of the presentinvention 1 mg/ml and human serum albumin 1 mg/ml divided in a vialcould maintain activity of the said compound for long term. Forculturing and activating cells in vitro, lyophilized preparation orliquid preparation of the polypeptide of the present invention areprepared and are added to the medium or immobilized in the vessel forculture. Toxicity of the polypeptide of the present invention wastested. Any polypeptide, 10 mg/kg was administered intraperitoneally inmice, but no death of mice was observed.

[0091] In vitro physiological activity of the polypeptide of the presentinvention can be evaluated by administering to disease model mice or itsresembled disease rats or monkeys, and examining recovery of physicaland physiological functions and abnormal findings. For example, in caseof searching abnormality in relation to hemopoietic cells. bone marrowsuppressive model mice are prepared by administering 5-FU series ofantitumor agents, and bone marrow cell counts, peripheral blood cellcounts and physiological functions are examined in the administeredgroup or the non administered group of mice. Further, in case ofsearching in vitro cultivation and growth of hemopoieticundifferentiated cells including hemopoietic stem cells, the bone marrowcells of mice are cultured in the groups with or without addition of thecompound of the present invention, and the cultured cells aretransferred into the lethal dose irradiated mice. Result of recovery isobserved with the indications of survival rate and variation of bloodcounts. These results can be extrapolated to the humans, and accordinglyuseful effective data for evaluation of the pharmacological activitiesof the compound of the present invention can be obtained.

[0092] Applications of the compound of the present invention forpharmaceuticals include diseases with abnormal differentiation of cells,for example leukemia and malignant tumors. These are cell therapy, whichis performed by culturing human derived cells in vitro with maintainingtheir original functions or adding new functions, and a therapy, whichis performed by regenerating without damaged the functions of theoriginally existed in the tissues by administering the compound of thepresent invention under the regeneration after tissue injury. Amount ofadministration may differ in the type of preparation and is ranged from10 μg/kg to 10 mg/kg.

[0093] Further strong physiological activity can be achieved byexpression of forming multimer of the polypeptide of the presentinvention.

[0094] Human Delta-2 having multimer structure can be produced by themethod of expressing chimera protein with human IgG Fc region asdescribed in the examples 3 and 4 and expressing the multimer havingdisulfide bond with hinge region of the antibody, or a method expressingchimera protein, in which antibody recognition region is expressed inthe C-terminal or N-terminal, and reacting with the polypeptidecontaining extracellular part of the thus expressed said human Delta-2and the antibody which recognize specifically the antibody recognitionregion in the C-terminal or N-terminal. In the other methods, a method,in which a fused protein with only the hinge region of the antibody isexpressed and the dimer is formed by disulfide bond, can be mentioned.The multimer of human Delta-2 having higher specific activity than thedimer can be obtained. The said multimer is constructed by fused proteinwhich is prepared for expressing the peptide in the C-terminal,N-terminal or other region. The protein is prepared in the form offorming disulfide bond without effecting in any activities of the otherhuman Delta-2. The multimer structure can also be expressed by arrangingone or more peptide, which is selected from polypeptides containingamino acids sequence of the sequence listing, SEQ ID NO: 1, 2 and 3,with genetic engineering method in series or in parallel. Other knownmethods for providing multimer structure having dimer or more can beapplied. Accordingly, the present invention includes any polypeptidescontaining amino acid sequences described in the sequence listing, SEQID NO: 1, 2 and 3 in the form of dimer or more structure prepared bygenetic engineering technique.

[0095] Further in the other method, multimerization method usingchemical cross-linker can be mentioned. For example,dimethylsuberimidate dihydrochloride for cross-linking lysine residue,N-(γ-maleimidebutyryloxy) succinimide for cross-linking thiol group ofcysteine residue and glutaraldehyde for cross-linking between aminogroups can be mentioned. The multimer with dimer or more can besynthesized by applying these cross-linking reactions. Accordingly, thepresent invention includes any polypeptides containing amino acidsequences described in the sequence listing, SEQ ID NO: 1, 2 or 3 in theform of dimer or multimer structure prepared by chemical cross-linkingagents.

[0096] In application of medical care in which cells are proliferatedand activated in vitro and are returned to the body, human Delta-2 ofthe form hereinabove can be added directly in the medium, butimmobilization can also be made. Immobilization method includes applyingamino group or carboxyl group in the peptide, using suitable spacers orthe above mentioned cross-linkers, and the polypeptide can be covalentlybound to the culture vessels. In example 11, method for preparation ofthe immobilized material and their effect are illustrated. Accordingly,the present invention includes any polypeptides containing amino acidsequences described in the sequence listing, SEQ ID NO: 1, 2 or 3 in theform of existing on the solid surface.

[0097] Since the natural human Delta-2 is cell membrane proteins,differentiation suppressive action in example 7, 8 and 12 can beexpressed by cocultivating with cells expressing these molecules andblood undifferentiated cells. Consequently, this invention includes amethod for cocultivation of undiferentiated cells with transformed cellsby using DNA coding amino acid sequence in the sequence listing, SEQ IDNO: 1, 2 or 3. An example is illustrated in example 10. Expressed cellmay be simian COS-7 cell or mouse Balb 3T3 cells as shown in examples,but cells of human origin are preferable, and further expressed cellsmay be any of human in vivo blood cells and somatic cells rather thancell lines. Consequently, the polypeptide can be expressed in vivo byintegrated into vectors for gene therapy. Examples of vectors for genetherapy are retrovirus vector, adenovirus vector or adeno-related virusvector.

[0098] This fact suggests that inhibition of binding of the polypeptidehaving amino acid sequence in the sequence listing, SEQ ID NO: 1, 2 or 3to these receptors can be used for finding out molecules and compoundsfor stimulating cell differentiation. The methods include bindingexperiment using radio isotope, luciferase assay using transcriptionalcontrol factors, a down stream molecule of the Notch receptor, andsimulation on the computer by X-ray structural analysis. Accordingly,the present invention includes a screening method for pharmaceuticalsusing polypeptide in the sequence listing, SEQ ID NO: 1, 2 or 3.

BRIEF EXPLANATION OF THE DRAWINGS

[0099]FIG. 1: Northern blotting analysis of expression of the humanDelta-2 mRNA in human organs.

[0100]FIG. 2: Binding of HD2EXIG of the present invention and of HD1EXIGas a control to the human origin T cell type cell line Jurkat.

[0101]FIG. 3: Binding of HD2EXIG of the present invention and of HD1EXIGas a control to CD34 positive cells from human umbilical cord bloodmononuclear cells.

EMBODIMENTS OF THE INVENTION

[0102] Following examples illustrate the embodiments of the presentinvention. but are not construed as limiting these examples.

REFERENTIAL EXAMPLE 1

[0103] Cloning of PCR Products Using Human Delta-1 Primer andDetermination of Base Sequence.

[0104] Mixed primers corresponding to amino acid sequence conserved inC-Delta-1 and X-Delta-1, i.e. sense primer DLTS1 (sequence listing, SEQID NO: 6) and antisense primer DLTA2 (sequence listing, SEQ ID NO: 7),were used.

[0105] A synthetic oligonucleotide was prepared by using automatic DNAsynthesizer with the principle of immobilized method. The automatic DNAsynthesizer used was 391PCR-MATE of Applied Biosystems Inc., U.S.A.Nucleotide, carrier immobilized with 3′-nucleotide, solution andreagents are used according to the instructions by the same corporation.Oligonucleotide was isolated from the carrier after finishing thedesignated coupling reaction and treating the oligonucleotide carrier,from which protective group of 5′-terminal was removed, withconcentrated liquid ammonia at room temperature for one hour. Forremoving the protective groups of nucleic acid and phosphoric acid, thereactant solution containing nucleic acid was allowed to stand in theconcentrated ammonium solution in the sealed vial at 55° C. for over 14hours. Each oligonucleotide, from which the carrier and protectivegroups were removed, was purified by using OPC cartridge of the AppliedBiosystems Inc., and detritylated by using 2% trifluoroacetic acid.Primer was dissolved in deionized water to set final concentration of100 pmol/μl for PCR after purification.

[0106] Amplification of these primers by PCR was performed as follows.Human fetal brain originated cDNA mixed solution (QUICK-Clone cDNA,CLONTECH Inc., U.S.A.) 1 μl was used. 10× buffer solution [500 mM KCl,100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, 0.01% gelatin] 5 μl, dNTP mixture(Takara Shuzo Co., Japan) 4 μl, sense primer DLTS1 (100 pmol/μl) 5 μlwhich was specific to the above vertebrates and antisense primer DLTA2(100 pmol/μl) 5 μl and TaqDNA polymerase (AmpliTaq, Takara Shuzo Co.,Japan, 5 U/μl) 0.2 μl were added thereto, and finally deionized waterwas added to set up total 50 μl. PCR was performed by 5 cycles of acycle consisting of treatment at 95° C. for 45 seconds, at 42° C. for 45seconds and 72° C. for 2 minutes, further 35 cycles of a cycleconsisting of treatment at 95° C. for 45 seconds, at 50° C. for 45seconds and 72° C. for 2 minutes, and finally allowed to stand at 72° C.for 7 minutes. A part of the PCR products was subjected to 2% agarosegel electrophoresis, stained with ethidium bromide (Nippon Gene Co.,Japan). and observed under ultraviolet light to confirm amplification ofabout 400 bp DNA.

[0107] Total amount of PCR product was subjected to electrophoresis with2% agarose gel prepared by low melting point agarose (GIBCO BRL Inc.,U.S.A.), stained by ethidium bromide, cutting out about 400 bp bands ofPCR products by the Delta primer under the UV light, adding distilledwater of the same volume of the gel, heating at 65° C. for 10 minutes,and completely dissolving the gel. The dissolved gel was centrifuged at15000 rpm for 5 minutes to separate supernatant solution after addingequal volume of TE saturated phenol (Nippon Gene Co., Japan) and thesame separation operation was performed after adding TE saturatedphenol:chloroform (1:1) solution and chloroform. DNA was recovered fromthe final solution by ethanol precipitation.

[0108] A vector, pCRII vector (Invitorogen Inc., U.S.A., hereinafterdesignates as pCRII) was used. The vector and the above DNA in molarratio of 1:3 were mixed and the DNA was ligated into the vector by usingT4 DNA ligase (Invitorogen Inc., U.S.A.). The pCRII, to which the DNAwas integrated, was subjected to gene transduction into E. coli one shotcompetent cells (Invitorogen Inc., U.S.A.) and was spread on thesemi-solid medium plate of L-Broth (Takara Shuzo Co., Japan) containingampicillin (Sigma Corp., U.S.A.) 50 μg/ml and allowed to stand at 37° C.for about 12 hours. The appeared colonies were randomly selected,inoculated in the L-Broth liquid medium 2 ml containing sameconcentration of ampicillin and shake cultured at 37° C. for about 18hours. The cultured bacterial cells were recovered and the plasmid wasseparated by using Wizard Miniprep (Promega Inc., U.S.A.) according tothe attached explanation sheet. The plasmid was digested by restrictionenzyme EcoRI. Integration of the said PCR product was confirmed byincision of about 400 bp DNA. Base sequence of the incorporated DNA inthe confirmed clone was determined by the fluorescent DNA sequencer(Model 373S. Applied System Inc., U.S.A.).

REFERENTIAL EXAMPLE 2

[0109] Cloning of Full Length Human Delta-1 and its Analysis A screeningof clones having full length cDNA was performed by hybridization fromhuman placenta origin cDNA library (inserted cDNA in λgt-11, CLONTECHInc., U.S.A.) in plaques corresponding to 1×10⁶ plaques. Generatedplaques were transferred onto nylon filter (HybondN+: Amersham Inc.,U.S.A.). The transcribed nylon filter was subjected to alkalinetreatment [allow to stand for 7 minutes on a filter paper permeated witha mixture of 1.5 M NaCl and 0.5 M NaOH], followed by twice neutralizingtreatments [allow to stand for 3 minutes on the filter paper permeatedwith a mixture of 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2) and 1 mM EDTA].Subsequently, the filter was shaken for 5 minutes in the 2-foldconcentrated SSPE solution [0.36 M NaCl, 0.02 M sodium phosphate (pH7.7) and 2 mM EDTA], washed and air-dried. Then the nylon filter wasallowed to stand for 20 minutes on the filter paper, which was permeatedwith 0.4 M NaOH, and was shaken for 5 minutes and washed with 5-foldconcentrated SSPE solution, and was then again air-dried. Screening wasconducted in the human Delta-1 probe labeled with radioisotope ³²P usingthese filters.

[0110] DNA probe prepared in referential Example 1 was labeled with ³²Pas follows. A DNA fragment was cut out by EcoRI from pCRII, inserted apurified PCR product (about 400 bp) by human Delta-1 primer anddetermined gene sequence, and was isolated from low melting pointagarose gel. The thus obtained DNA fragment was labeled by DNA labelingkit (Megaprime DNA labeling system: Amersham, U.S.A.). The primersolution 5 μl and deionized water were added to the DNA 25 ng to set uptotal volume of 33 μl, which was treated for 5 minutes in boiling waterbath. Reaction buffer solution 10 μl containing dNTP, α-³²P-dCTP 5 μland T4 DNA polynucleotide kinase solution 2 μl were added thereto,treated at 37° C. for 10 minutes in water bath. Subsequently, themixture was purified by Sephadex column (Quick Spin Column SephadexG-50: Boehringer Mannheim Inc., Germany), then treated for 5 minutes inboiling water bath and ice-cooled for 2 minutes for use.

[0111] Hybridization was performed as follows. The prepared filterhereinabove was immersed into the prehybridization solution consistingof SSPE solution, a final concentration of each component of which isset at 5-fold concentration, 5-fold concentration of Denhardt's solution(Wako Pure Chemicals, Japan), 0.5% SDS (sodium dodecyl sulfate, WakoPure Chemicals, Japan) and salmon sperm DNA (Sigma, U.S.A.) 10 μg/mldenatured by boiling water, and shaken at 65° C. for 2 hours, then thefilter was immersed into the hybridization solution of the samecomposition of the above prehybridization solution containing³²P-labeled probe by the above described method and shaken at 65° C. for16 hours to perform hybridization.

[0112] The filter was immersed into SSPE solution containing 0.1% SDS,shaken at 55° C. and washed twice, further immersed into 10-folddilution of SSPE solution containing 0.1% SDS and washed four times at55° C. An autoradiography of the washed filter was performed usingintensified screen. Clones of strongly exposed part were collected andthe plaques obtained were again spread and screened by the same methodhereinbefore to separate complete single clones.

[0113] The thus isolated phage clones were seven clones. Phage of all ofthese clones was prepared to about 1×10⁹ pfu, purified the phage DNAusing Withered Lambda Rep (Promega Corp., U.S.A.), digested byrestriction enzyme EcoRI and inserted into pBluescript KS (StratageneInc., U.S.A.), which was digested by EcoRI in the same way. DNAsequences of the both ends of these clones were analyzed by DNAsequencer. Three clones of D5, D6 and D7 were the clone containing DNAsequence from No. 1 to 2244 in the sequence listing, SEQ ID NO: 8. Aclone D4 was a clone containing DNA sequence from No. 999 to 2663 in thesequence listing, SEQ ID NO: 8. The clones D5 and D4 prepared thedeletion mutant by using kilosequence deletion kit (Takara Shuzo Co.,Japan) according to a description of the attached paper, Full-lengthcDNA base sequence of the present invention was determined using the DNAsequencer from both direction of 5′-direction and 3′-direction.

[0114] By applying with XhoI site at No. 1214 in DNA sequence in thesequence listing, SEQ ID NO: 8, D4 and D5 were digested by restrictionenzyme XhoI to prepare plasmid pBSDel-1 containing full length of DNAsequence in the sequence listing, SEQ ID NO: 8.

EXAMPLE 1

[0115] Cloning of cDNA of the Novel Human Delta-2

[0116] Gene cloning of new human Delta homologue was performed usingprobe of the human Delta-1 gene in the sequence listing, SEQ ID NO: 8.

[0117] Probe was prepared by PCR using a template of the human Delta-1full length gene pBSDel-1 in the sequence listing, SEQ ID NO: 8 obtainedin the referential example 2, or vector pUCDL-1F, which was deposited inthe above described institution. A sense primer in the sequence listing,SEQ ID NO: 9 (DNA sequence corresponding to a sequence from No. 636 to655 in the sequence listing, SEQ ID NO: 8) and antisense primer in thesequence listing, SEQ ID NO: 10 (a complementary chain of DNA sequencecorresponding to a sequence from No. 1332 to 1351 in the sequencelisting, SEQ ID NO: 8) were used as primers.

[0118] A composition of solution for amplification by PCR was acomposition of solution as described in the referential example 1,except for the primer and the template. PCR was conducted by thefollowing conditions: a cycle consisting of a cycling at 95° C. for 45seconds, at 55° C. for 45 seconds and at 72° C. for 2 minutes, a cycleof which was performed for 30 cycles, and finally the mixture was allowto stand at 72° C. for 7 minutes. A part of the PCR product wassubjected to 1% agarose gel electrophoresis, stained with ethidiumbromide (Nippon Gene Corp., Japan) and observed by UV light to confirmamplification of about 700 bp cDNA.

[0119] The PCR product was cut out from the agarose gel, and the DNAprobe was purified according to a description of the attached sheet inGeneclean II kit (Bio101 Corp., U.S.A.) and diluted with distilled waterto 25 ng/μl to prepare DNA probe having sequence shown in the sequencelisting, SEQ ID NO: 11.

[0120] A human fetal lung cDNA library prepared by λgt10 (ClontechCorp., U.S.A.) was screened using the above probe according to a methoddescribed in the referential example 2. In the screening, ahybridization was conducted at 55° C. for 16 hours, with washingcondition for immersing into SSC solution containing 1% SDS, shaking atroom temperature and washing six times. Further, a condition immersinginto SSC solution, which was diluted 3-fold containing 0.1%SDS, andwashing at 55° C. was performed.

[0121] About 120 million plaques were screened in the first screeningunder the above condition, and as a result, about 120 plaques determinedas positive, were subjected to the second screening by the similarmethod to separate each phage.

[0122] The isolated phage DNA was purified according to a method in thereferential example 2, digested by restriction enzyme EcoRI, ligated topBluescript KS and analyzed DNA sequence by DNA sequencer by the samemethod as described in the referential examaple 1.

[0123] About over half number of clones was human Delta-1 having genesequence in the sequence listing, SEQ ID NO: 8. Among these, fiveclones, which were similar to but have different gene sequence of thehuman Delta-1, containing new sequences, which were not found in GenbankRelease 98 by computer software Genetyx CD Ver 36 (Software DevelopmentCorp.) were found. Deletion mutant of DNA sequence of these clones wasprepared using kilosequence deletion kit (Takara Shuzo Co., Japan)according to the attached instruction manual. Base sequence of fulllength cDNA of the present invention was determined from both directionof 5′-direction and 3′-direction by combining with primer walking methodusing the said DNA sequencer.

[0124] As a result, clone 4A encodes gene sequence from No. 526 to 3339of DNA sequence in the sequence listing, SEQ ID NO: 4 (proviso thatsequence from No. 1296 to 1515 was deleted); clone 22 encodes genesequence from No. 1029 to 3213 of DNA sequence in the sequence listing,SEQ ID NO: 4; clone 65 encodes gene sequence from No. 754 to 3228 of DNAsequence in the sequence listing, SEQ ID NO: 4; clone 90 encodes genesequence from No. 552 to 2618 of DNA sequence in the sequence listing,SEQ ID NO: 4; and clone 105 encodes gene sequence from No. 669 to 3339of DNA sequence in the sequence listing, SEQ ID NO: 4 (proviso that inthe clone 105, since many insertions of unknown sequences, which werenot found in the other clones, were observed in many regions, it mightbe originated from prior splicing immature mRNA). In addition, in theclone 65, cytosine of DNA sequence No. 2294 in the sequence listing, SEQID NO: 4 was replaced by thymine. Therefore, serine of DNA sequence No.647 in the sequence listing was replaced by threonine.

[0125] These clones did not, however. contain gene sequence coding fulllength amino acid sequence, further new probe was prepared and screeningwas repeated.

[0126] New probe was prepared by PCR using template of clone 4A whichwas isolated in the above process. A probe was prepared by using senseprimer of the sequence listing, SEQ ID NO: 12 (which corresponds to asequence from No. 526 to 545 of DNA sequence in the sequence listing,SEQ ID NO: 4) and antisense primer of the sequence listing, SEQ ID NO:13 (which corresponds to a sequence from No. 918 to 937 of DNA sequencein the sequence listing, SEQ ID NO: 4), and by the same way as describedpreviously. DNA sequence of this probe is shown in the sequence listing,SEQ ID NO: 14.

[0127] The screening of the cDNA library using the probe was conductedas same in the first screening procedure. Proviso that the hypridizationwas performed at 65° C. for 16 hours; washing was performed by immersinginto SSC solution containing 0.1% SDS with shaking at room temperature,further immersing into 10-fold diluted SSC solution containing 0.1% SDSwith washing twice at 65° C.

[0128] New clones were identified by the screening. As a result of genesequence determination, the clone P having identical sequence with DNAin the sequence listing, SEQ ID NO: 4, and the clone RA having DNAsequence from No. 263 to 2768 in the sequence listing, SEQ ID NO: 4 weredetermined. These two clones were isolated and identified as clonescoding full length novel human Delta-2 protein. A vector containingclone P, which is ligatedin EcoRI site of pBluescript KS, is designatedas pBSDL-2.

EXAMPLE 2

[0129] Organs Which Express Novel Human Delta-2

[0130] In order to search expression of the new human Delta-2 mRNA,using filters of Human Multiple Tissue Northern Blot, Human MultipleTissue Northern Blot II, Human Multiple Tissue Northern Blot III andHuman Fetal Multiple TissueNorthern Blot II (Clontech Corp., U.S.A.),for which mRNA was previously transcripted, and using DNA havingsequence of the sequence listing, SEQ ID NO: 14 described in the example1 as a probe, ³²P labeling was performed by the above mentionedprocedure using the previously mentioned DNA labeling kit (Megaprime DNAlabeling system: Amersham Corp., U.S.A.), hybridization was conductedaccording to the instruction manual attached to the above filters todetect the expression. Result is shown in FIG. 1.

[0131] As a result, two types of the expressed mRNA having length about3.8 kb and 5 kb were found. The most strong expression in human adulttissues as expression site was the heart. Relatively strong expressionswere observed in the placenta, ovary, small intestine, thyroid gland andspinal cord. Obvious expressions were observed in the skeletal muscle,lung, liver, pancreas, thymus, prostate, lymph node, trachea, adrenalgland and bone marrow. Extremely weak expressions were observed in thestomach, spleen and colon. Further no expressions were observed in thebrain, kidney, testis and peripheral blood lymphocyte. Highly expressionwas observed in the fetal lung among the human fetal tissues. Strongexpression is observed in the fetal kidney and weak expressions wereobserved in the fetal liver and fetal brain.

[0132] These results indicate that the novel human Delta-2 of thepresent invention might have function relating to the heart in adult.Further, it might have function against vascular cells due to finding ofexpression in the fetal lung.

EXAMPLE 3

[0133] Preparation of Expression Vectors of the Novel Human Delta-2

[0134] Using the vector pBSDL-2 coding the full length novel humanDelta-2 in example 1, the expression vectors of human Delta-2 proteinsmentioned in the following 1) -5) were prepared. Addition of restrictionenzyme sites and insertion of short gene sequence were performed usingExSite PCR-Based Site-Directed Mutagenesis Kit (Stratagene Inc., U.S.A.)according to the instruction manual.

[0135] 1) Expression Vector of Secretory Novel Human Delta-2 Protein(HD2EX)

[0136] The cDNA coding polypeptide having amino acid sequence from No. 1to 500 in the sequence listing, SEQ ID NO: 2 was ligated into theexpression vector pcDNA3 containing cytomegalovirus promoter andneomycin resistance gene to prepare the expression vector.

[0137] For preparation of expression vector of the novel human Delta-2,in order to make stable expression of gene product, EcoRI site was addedin the 20 bp upper stream for 5′-direction of the initiation codon (genesequence No. 277 in the sequence listing, SEQ ID NO: 4). Using the aboveMutagenesis Kit, a plasmid pBSDL-2, which contained DNA sequence insequence listing, SEQ ID NO: 4 and full length cDNA of human Delta-2were set as the template, and oligonucleotides having gene sequences insequence listing, SEQ ID NO: 15 and SEQ ID NO: 16, ware set as theprimers. Then DNA adding EcoRI site in the 20 bp upper stream for5′-direction was prepared. Hereinafter this plasmid is designated aspBSEco-DL-2.

[0138] Next, in order to add the termination codon and restrictionenzyme NotI site after the extracellular C-terminal position, i.e. afterDNA sequence coding amino acid sequence up to No. 500 serine residue inthe sequence listing, SEQ ID NO: 2, by using the Mutagenesis Kitsimilarly, using the pBSEco-DL-2 as template and settingoligonucleotides having gene sequences in the sequence listing, SEQ IDNO: 17 and SEQ ID NO: 18 as primers, addition of the termination codonand NotI site were performed. Then, the resulted vector was digested byEcoRI and NotI, and about 1600 bp split gene fragment was ligated inpcDNA3, which was treated by the same restriction enzymes, to constructthe expression vector. This vector was designated as pHD2EX.

[0139] 2) Expression Vector of FLAG Chimera Protein of Secretory NovelHuman Delta-2 (HD2EXFLAG)

[0140] The cDNA coding chimera protein having amino acid sequence fromNo. 1 to 500 in the sequence listing, SEQ ID NO: 2 and at thisC-terminal the FLAG sequence was ligated to the expression vector pcDNA3to prepare the expression vector.

[0141] Using pBSEco-DL-2 as template, FLAG sequence was added in theextracellular C-terminal, i.e. after serine residue at No. 500 aminoacid in the sequence listing, SEQ ID NO: 2. Then, in order to add thetermination condon and restriction enzyme NotI site, using theMutagenesis Kit, and setting oligonucleotides having gene sequences inthe sequence listing, SEQ ID NO: 19 and SEQ ID NO: 18 as primers, a genecoding FLAG sequence, termination codon and NotI site were added in theC-terminal. This vector was digested by EcoRI and NotI, and about 1600bp split gene fragment was ligated to pcDNA3 treated similarly by therestriction enzyme to construct the expression vector. This vector wasdesignated as pHD2EXFLAG.

[0142] 3) Expression Vector of IgG1Fc Chimera Protein of Secretory HumanDelta-2 (HD2EXIg)

[0143] A gene sequence coding polypeptide having amino acid sequence inthe sequence listing, SEQ ID NO: 2 and at this C-terminal an amino acidsequence of Fc region downstream from the hinge region of human IgG1,was ligated into the expression vector pcDNA3 to prepare the expressionvector.

[0144] Preparation of fused protein with immunoglobulin Fc protein wasperformed according to the method of Zettlmeissl et al. (Zettlmeissl etal., DNA cell Biol., 9, 347-354, 1990). A gene using genome DNA withintron was applied and the said gene was prepared by using PCR.

[0145] Human genome DNA was used as a template and genomic gene sequencecoding human IgG1Fc region was subjected to PCR by using primers of anoligonucleotide having the sequence in the sequence listing. SEQ ID NO:23 with restriction enzyme BamHI site and an oligonucleotide having thesequence in the sequence listing, SEQ ID NO: 24 with restriction enzymeXbaI site. The thus obtained about 1.4 kbp band was purified anddigested by restriction enzyme BamHI and XbaI (Takara Shuzo Co., Japan),and genes were ligated to pBluescript, which was similarly treated byrestriction enzyme, by using T4 DNA ligase to perform subcloning.

[0146] Later, the plasmid DNA was purified and confirmed gene sequenceusing sequencer, then the said gene sequence was confirmed as genomicDNA in the hinge region of heavy chain of the human IgG1. (The sequenceis referred to Kabat et al., Sequence of Immunological Interest, NIHPublication No.91-3242,1991). Namely, this gene has the restrictionenzyme BamHI site at 5′-terminal and XbaI site at 3-terminal, and iscloned with BamHI site and XbaI site of pBluescript KS. Hereinafter thisplasmid is designated as pBShIgFc.

[0147] Using the pBSEco-DL-2 as template, and using the Mutagenesis Kit,restriction enzyme BamHI site was added in the extracellular C-terminal,i.e. after serine at No. 500 in the sequence listing, SEQ ID NO: 3.Furthermore, in order to add restriction enzyme NotI site to thedownstream for ligating DNA coding the above human immunoglobulinIgG1Fc, these sites were added by using oligonucleotides having genesequences in the sequence listing, SEQ ID NO: 20 and SEQ ID NO: 18 andthe Mutagenesis Kit. During this process, in order to not to shift theframe coding amino acid as a result of addition of BamHI site, AGC inthe DNA sequence coding No. 500 serine on the DNA sequence in thesequence listing, SEQ ID NO: 4, was replaced to TCG.

[0148] The thus prepared vector was digested by NotI and BamHI, andabout 1200 bp gene fragment digested and splited from the above pBShIgFcby NotI and BamHI was ligated into the digested vector to preparefinally the vector containing gene fragments coding the objectivesecretory human Delta-2 IgG1Fc chimera protein. Finally, this vector wasdigested by EcoRI and NotI and about 3000 bp split gene fragment wasligated into pcDNA3 which was treated similarly with the restrictionenzymes, to construct the expression vector. This vector was designatedas pHD2EXIg.

[0149] 4) Expression Vector of Full Length Human Delta-2 Protein (HD2F)

[0150] The cDNA coding polypeptide from No. 1 to 659 of amino acidsequence in the sequence listing, SEQ ID NO: 4, was ligated into theexpression vector pcDNA3 to prepare the expression vector.

[0151] In order to add the termination codon and restriction enzyme NotIsite in C-terminal of the full length sequence, i.e. after Val at No.659 in the sequence listing, SEQ ID NO: 3 by using pBSEco-DL-2 astemplate, and using the Mutagenesis kit similarly oligonucleotideshaving gene sequences in the sequence listing, SEQ ID NO: 21 and SEQ IDNO: 18 were set as primers, and the termination codon and NotI site wereadded in the C-terminal. This vector was digested by EcoRI and NotI, andabout 2200 bp split gene fragment was ligated into pcDNA3, which wastreated similarly by restriction enzymes, to construct the expressionvector. This vector was designated as pHD2F.

[0152] 5) Expression Vector of FLAG Chimera Protein (HD2FLAG) of FullLength Novel Human Delta-2

[0153] The cDNA coding chimera protein, having an amino acid sequencefrom No. 1 to 659 in the sequence listing. SEQ ID NO: 3 and at thisC-terminal the FLAG sequence, was ligated into the expression vectorpcDNA3 to prepare the expression vector.

[0154] In order to add FLAG sequence, the termination codon andrestriction enzyme NotI site in the C-terminal, by using pBSEco-DL-2 astemplate, oligonucleotides having gene sequences in the sequencelisting, SEQ ID NO: 22 and SEQ ID NO: 18 were used as primers, and agene coding FLAG sequence and termination codon as well as NotI sitewere added in the C-terminal.

[0155] This vector was digested by EcoRI and NotI, and about 2100 bpsplit gene fragments were ligated into pcDNA3, which was treatedsimilarly by restriction enzyme to construct the expression vector. Thisvector was designated as pHD2FLAG.

EXAMPLE 4

[0156] Gene Transfer of Expression Vectors Into Cells and Expression

[0157] The expression vectors prepared in Example 3 were transferredinto COS-7 cell (obtained from RIKEN Cell Bank, Physical and ChemicalResearch Institute, Japan, RCB0539).

[0158] Cell culture before gene transfer was performed by culturing inD-MEM (Dulbecco modified Eagle's medium, GIBCO-BRL Inc., U.S.A.) 10%FCS. On a day before gene transfer, medium of cells was changed to setcell counts 5×10⁵ cells/ml and cultured for overnight. On the day ofgene transfer, cells were sedimented by centrifugation, washed twicewith PBS (−) by centrifuge and prepared to 1×10⁷ cells/ml in 1 mM MgCl₂and PBS (−). Gene transfer was performed by electroporation using genetransfer device Gene-pulsar (BioRad Inc., U.S.A.). The above cellsuspension 500 μl was collected in a cell exclusively forelectroporation (0.4 cm), added expression vector 20 μg, and allowed tostand on ice for 5 minutes. Thereafter, voltage was charged twice underthe condition of 3 μF. 450 V, and between the two charges the cellmixture was allowed to stand at room temperature for 1 minute. After 5minutes allowed to stand on ice, cells were spread in the culturemedium, diameter 10 cm previously added 10 ml of medium, and cultured at37° C. in 5% carbon dioxide incubator.

[0159] Next day, the cultured supernatant solution was removed, washedthe cells adhered to the dish twice with PBS(−) 10 ml. In cases ofexpression vectors pHD2EX, pHD2EXFLAG and pHD2EXIg, serum-free D-MEM 10ml was added and cultured for further 7 days. Cultured supernatantsolution was recovered and was replaced the buffer to PBS (−) byCentricon 30 (Amicon Inc., U.S.A.) and simultaneously the solution wasconcentrated to 10-fold to obtain cell cultured supernatant solution.

[0160] In cases of pHD2F and pHD2FLAG, medium was changed by D-MEMcontaining 10% FCS, and cultured further 3 days to prepare cell lysate.Thus, 2×10⁶ cells were suspended in the cell lysis buffer [50 mM Hepes(pH 7.5), 1% Triton X100, 10% glycerol, 4 mM EDTA, 50 μg/ml Aprotinin,100 μM Leupeptin, 25 μM Pepstatin A and 1 mM PMSF] 200 μl, allowed tostand on ice for 20 minutes and centrifuged at 14000 rpm for 20 minutesto remove supernatant solution to obtain cell lysate.

[0161] Using the thus obtained samples, expression of proteins weredetected by Western blotting.

[0162] Namely, concentrated cultured supernatants or cell lysates weresubjected to SDS-PAGE using an electrophoresis tank and polyacrylamidegel for SDS-PAGE (gradient gel 5-20%) (ACI Japan Inc., Japan) accordingto the attached instruction manual. Samples were prepared by treatmentin boiling water for 5 minutes with 2-mercaptoethanol (2-ME) forreduction, and non-reduced condition without taking the above treatment.As a markers Rainbow Marker (higher molecular weight, Amersham Inc.) wasused. Sample buffer solution and electrophoresis buffer were preparedwith reference to the attached leaflet. When the SDS-PAGE was finished,acrylamide gel was transcribed to PVDF membrane filter (BioRad Inc.,U.S.A.) using the Mini Trans Blot Cell (BioRad Inc.).

[0163] The thus prepared filter was shaken overnight at 4° C. in theBlockace (Dainippon Pharm. Co., Japan) or TBS-T [20 mM Tris, 137 mM NaCl(pH 7.6) and 0.1% Tween 20] containing 5% bovine serum albumin (SigmaCo., U.S.A.) for blocking. Thereafter, according to the explanation ofthe attached leaflet of the ECL Western blotting detection system(Amersham Inc., U.S.A.); anti-human Delta-2 mouse monoclonal antibodydescribed in Example 6 or mouse monoclonal antibody Anti-FLAG M2 (KodakInc. U.S.A.) for FLAG chimera (HD2EXFLAG and HD2FLAG) was used asprimary antibody and peroxidase labeled anti-mouse Ig sheep antibody(Amersham Corp., U.S.A.) was reacted as secondary antibody. In case ofIgG chimera, peroxidase labeled anti-human Ig sheep antibodies (AmershamInc., U.S.A.) was reacted.

[0164] Reaction time for antibodies was 1 hour at room temperature, andat an interval of each reaction, washing was performed by shaking inTBS-T at room temperature for 10 minutes for three times. After thefinal washing, the filter was immersed in the reaction solution ofECL-Western blotting detection system (Amersham Inc., U.S.A.) for 5minutes, and wrapped in polyvinylidene chloride wrapping film to exposeX-ray film.

[0165] As the result, in the sample with treatment of reduction, thebands showing a protein obtained by transfer of pHD2EX and pHD2EXFLAGwere detected a band of about 65 kD and a protein obtained by transferof pHD2EXIg was detected a band of about 95 kD. In the non-reducedsample, the bands showing protein obtained by transfer of pHD2EXIg weredetected slightly smeared bands at 150 kD to 200 kD, mainly about 180kD, which showed about 2-fold of the reduction stage, consequently,dimer was formed.

[0166] In these experiments, however, although cell lysate and culturedsupernatant of COS-7 cells, to which pcDNA3 vector was transferred as acontrol, were tested, no bands reacted against anti-human Delta-2 mousemonoclonal antibody, anti-FLAG antibody, and anti-human Ig antibody weredetected.

[0167] Therefore, these five expression vectors can produce theobjective polypeptides.

EXAMPLE 5

[0168] Purification of Secretory Novel Human Delta-2 Proteins of GeneTransfer Cells

[0169] Cultured supernatants of COS-7 cells containing HD2EXFLAG orHD2EXIg, both of which expression was detected by the method in example4, were prepared in large scale, and each chimera protein was purifiedby affinity column.

[0170] In case of HD2EXFLAG, 2 liter of the cultured supernatantobtained by the method in the example 4 was passed through a columnpacked with Anti-FLAG M2 Affinity Gel (Eastman Kodak, U.S.A.) and thechimera protein was adsorbed in a column by an action of affinitybetween FLAG sequence of the chimera protein and Anti-FLAG antibody ofthe gel. Column, a disposable column (BioRad Inc., U.S.A.) with innerdiameter of 10 mm, was used with packing the above gel 5 ml. Acirculation system consisting of medium bottle→column→peristalticpump→medium bottle was set up. The circulation was run by a flow 1ml/min. for 72 hours. Thereafter the column was washed with PBS (−) 35ml and eluted with 0.5 M Tris-glycine (pH 3.0) 50 ml. The eluate of 25fractions, each 2 ml, was collected into the tube, and each fraction wasneutralized by 200 μl of 0.5 M Tris-HCl (pH 9.5) previously added ineach tube.

[0171] The eluate fraction, each 10 μl of the secretory FLAG chimeraprotein, which was purified by the above method, was subjected toreduction treatment described in Example 4. SDS-PAGE electrophoresis by5-10% gradient polyacrylamide gel was performed. After finishing theelectrophoresis, silver staining was conducted by using Wako silverstain kit 11 (Wako Pure Chemicals, Japan) according to the explanationof the attached leaflet. As a result, a band of HS2EXFLAG was detectedin the eluate fractions from No. 4 to 8. Molecular weight thereof wasidentical with the result of Western blotting of anti-FLAG antibodyobtained in the example 4. Therefore, purified HD2EXFLAG was obtained.

[0172] In the IgG1Fc chimera protein, i.e. HD2EXIg, 2 liters of thecultured supernatant was adsorbed to Protein A Sepharose column(Pharmacia Inc., Sweden) according to the same method as of FLAG chimeraprotein to collect the eluate fractions. Using a part of eluate as sameas in FLAG chimera protein, a determination of the eluate fraction,identification of the size and detection of the purity were performed bySDS-PAGE electrophoresis and silver staining in the reduced condition.As a result, bands were detected in the eluate fraction from No. 4 to15. The size thereof is identical with the result of Western blottingusing anti-human Ig. Therefore, purified HD2EXIg was obtained.

[0173] Molecular weight of the thus purified HD2EXFLAG was furtheranalyzed in details by SDS-PAGE. The molecular weight was confirmed astwo bands, one of which was 65.8 KD and the other was 61.7 KD. Thesedifferent two bands having different molecular weights were transcribedinto PVDF according to the method in the example 4, and ten amino acidsof N-terminal amino acid sequence were determined by amino acidsequencer (ABI Corp., U.S.A.). As a result, each amino acid sequence wasidentical with the amino acid sequence from No. 1 to 10 in the sequencelisting, SEQ ID NO: 1. This result indicates that difference inmolecular weight might be due to difference in attached sugar chain.Similarly, in the purified HD2EXIg, two bands having slightly differentmolecular weight were confirmed and were thought to be due to the samereason.

EXAMPLE 6

[0174] Preparation of Antibody Recognizing Novel Human Delta-2

[0175] HD2EXFLAG, purified by the method in Example 5, was used asimmunogen, and rabbits were immunized. After assaying antibody titer,whole blood was collected and serum was obtained. Anti-human Delta-2rabbit polyclonal antibody was prepared by using the Econopack serum lgGpurification kit (BioRad Inc., U.S.A.) with reference to the attachedinstruction manual.

[0176] HD2EXFLAG purified by a method described in the example 5 wasused as immunogen, and mouse monoclonal antibody was prepared accordingto the explanation in the textbook. The purified HD2EXFLAG wasadministered in Balb/c mice (Nippon SLC CO., Japan), 10 μg/mouse,immunized intracutaneously and subcutaneously. After secondimmunization, increased serum titer was confirmed by collecting bloodophthalmologically, the third immunization was performed. Subsequently,the spleens of mice were collected and fused with mouse myeloma cellsP3X63Ag8 (ATCC TIB9) using polyethylene glycol. Hybridoma was selectedby HAT medium (Immunological and Biological Research Institute, Japan),and the hybridoma strains, which produced antibody specificallyrecognizing extracellular region of novel human Delta-2 in the medium,were isolated by enzyme immunoassay. The hybridoma strains producingmouse monoclonal antibody, which specifically recognized novel humanDelta-2, were established.

[0177] The novel human anti-human Delta-2 monoclonal was purified andprepared by using Mab Trap GII (Pharmacia Inc., Sweden) according to theexplanation of the attached instruction manual from the supernatant ofthe thus established hybridoma.

[0178] Using the monoclonal antibodies, affinity column was prepared.Preparation of the affinity column was performed according to theinstruction manual attached to the CNBr activated Sephadex 4B (PharmaciaInc., Sweden). A column, 2 cm²×1 cm, containing gel 2 ml, was prepared.A concentrated solution of the supernatant of the cultured COS-7 cells,to which pHD2EX was gene transferred, was passed through the column, forwhich anti-human Delta-2 monoclonal antibody was bound, at 20 ml/hr,subsequently PBS (−) 15 ml was passed at the same flow rate and washedthe column. Finally, the product was eluted by a mixture of 0.1 M sodiumacetate and 0.5 M NaCl (pH 4.0). The eluate, each 1 ml fraction, wascollected, and was neutralized by adding 1M Tris-HCl (pH 9.1) 200 μl foreach fraction.

[0179] SDS-PAGE of each purified protein was conducted under reducedcondition according to the method described in the example 4, followedby silver staining and Western blotting to estimate molecular weight. Asa result, HD2EX of about 65 kD was purified from concentratedsupernatant of the cultured COS-7 cells, to which pHD2EX was genetransferred. Consequently, the novel human Delta-2 protein can bepurified by the affinity column.

EXAMPLE 7

[0180] Effects of HD2EXIg to Colony Formation of Blood UndifferentiatedCells

[0181] In order to observe physiological action of HD2EXIg on bloodundifferentiated cells, CD34 positive cells were cultured in theserum-free semi solid medium in the presence of HD2EXIg and knowncytokines, and number of colony forming cells were observed.

[0182] Human umbilical cord blood or adult human normal bone marrowblood was treated by the silica solution (Immunological and BiologicalResearch Institute, Japan) according to the attached instruction manual,then the low density cellular fraction (<1.077 g/ml) was fractionated bydensitometric centrifugation of Ficoll pack (Pharmacia Inc., Sweden) toprepare mononuclear cells, and CD34 positive cells of human umbilicalcord blood or human normal bone marrow blood was isolated from themononuclear cells.

[0183] Separation of CD34 positive cells was performed by usingDynabeads M-450 CD34 and DETACHaBEADS CD34 (Dynal Inc., Norway)according to attached instruction manual. After separation, the puritywas measured as follows. Cells were stained by FITC labeled CD34antibody HPCA2 (Beckton-Deckinson Inc., U.S.A.) and examined byflow-cytometer (FACSCalibur, Beckton-Deckinson, U.S.A.). Purity above85% was confirmed for use.

[0184] The thus isolated CD34 positive cells were suspendedhomogeneously to form 400 cells/ml of the medium hereinbelow, and spreadin the 35 mm dish (Falcon Inc., U.S.A.), then cultured for 2 weeks incarbon dioxide incubator at 37° C. under 5% carbon dioxide, 5% oxygen,90% nitrogen and 100% humidity. The thus formed blood colonies werecounted under the invert microscope.

[0185] A medium used is α-medium (GIBCO-BRL, U.S.A.), containing 2%deionized bovine serum albumin (BSA, Sigma, U.S.A.) 10 μg/ml humaninsulin (Sigma, U.S.A.), 200 μg/ml transferrin (Sigma, U.S.A.), 10⁻⁵M2-mercaptoethanol (Nakarai Tesk Co., Japan), 160 μg/ml soybean lecithin(Sigma, U.S.A.), 96 μg/ml cholesterol (Sigma, U.S.A.) and 0.9%methylcellulose (Wako Pure Chemicals, Japan).

[0186] To the above medium, the novel human Delta-2 extracellular Igchimera protein (HD2EXIg) was added to the final concentration of 1μg/ml. For control, human IgG1 (Athens Research and Technology Inc.,U.S.A.) was added with the same concentration in order to observe effectof IgGFc region.

[0187] Conditions of simultaneously added cytokines were as follows: 100ng/ml human SCF (Intergen Inc., U.S.A.), 10 ng/ml human IL-3 (IntergenInc., U.S.A.), and 100 ng/ml human IL-6 (Intergen Inc., U.S.A.).

[0188] As a result. in the control group, number of colony formation was42±5 per 400 cells, and in HD2EXIg added group, number of colonyformation was 21±3, which showed significant suppression of colonyformation. Result indicates that the novel human Delta-2 of the presentinvention has an action on the blood undifferentiated cells.

EXAMPLE 8

[0189] Action of HD2EXIg on LTC-IC of Blood Undifferentiated Cells inSerum Free Liquid Culture

[0190] For confirmation of physiological action of HD2EXIg on theundifferentiated blood cells in the liquid culture, umbilical cord bloodmononuclear CD34 positive cells were cultured in serum-free medium inthe presence of HD2EXIg and known cytokines. Culture has continued for 2weeks, and at the present day on 2 weeks culture. changes of LTC-IC,which was thought to be most undifferentiated blood cells. wereconfirmed.

[0191] For comparative studies. a control experiment without addingHD2EXIg and an experiment with addition of HD1EXIg, which was IgGchimera protein (HD1EXIg) and was found to have LTC-IC activity in thesimilar experiment disclosed in WO 97/19172 in the name of presentinventors, were conducted. Preparation of HD1EXIg was performedaccording to the description of WO 97/19172.

[0192] Sixteen thousands and two hundreds CD34 positive cells ofumbilical cord blood mononuclear cells isolated by a method described inthe example 7 were cultured in the following medium. Numbers of LTC-ICwere counted in the four experimental groups of the pre-culture group,HD2EXIg added group, HD1EXIg added group and control group. Number ofcells and number of colony forming cells were also counted.

[0193] Culture was performed in the basal medium of a-medium, to which2% BSA, 10 μg/ml human insulin, 200 μg/ml transferrin, 40 μg/ml lowdensity lipoprotein, 10⁻⁵M 2-mercaptoethanol, 100 ng/ml human SCF, 10ng/ml human IL-3 and 100 ng/ml human IL-6 were added. To the HD2EXIgadded group, purified HD2EXIg 1 μg/ml was added; to the HD1EXIg addedgroup, purified HD1EXIg 1 μg/ml was added; and to the control group, theabove human IgG1 was added. Exchange of medium was conducted twice aweek with changing half volume.

[0194] LTC-IC was measured according to a method of Sutherland et al.(Blood, 74, 1563-, 1989; Proc. Natl. Acad. Sci., U.S.A., 87, 3584-,1990). In details, refer to WO 97/18172 in the name of the presentinventors.

[0195] Total cell counts were measured by counting numbers of livingcells microscopically using trypan blue (Gibco BRL, U.S.A.). Numbers ofcolony forming cells were performed according to a method in the example7 using the following medium. The medium is a-medium, to which 30% fetalcalf serum (FCS, ICN Biomedical Japan, Japan), 1% BSA, 10⁻⁵ M2-mercaptroethanol, 0.9% methylcellulose (Wako Pure Chemicals, Japan),100 ng/ml human SCF, 10 ng/ml human IL-3, 100 ng/ml human IL-6, 2U/mlhuman EPO (Chugai Seiyaku Co., Japan) and 10 ng/ml human G-CSF(IntergenInc., U.S.A.).

[0196] Result is shown in Table 1. TABLE 1 Effect of the human Delta-2of the present invention on liqid culture Total cell counts Colonyforming cells LTC-IC Pre-culture group 16200 2500 150 Post-culture groupControl 445000 23000 3.3 HD1EXIg 395000 16700 9.3 HD2EXIg 418000 1600016.2

[0197] Result indicates that HD2EXIg has maintenance activity fornumbers of LTC-IC as compared with the control group. Further. theactivity is stronger than HD1EXg.

EXAMPLE 9

[0198] Binding and Separation of HD2EXIg for Blood UndifferentiatedCells.

[0199] Binding of purified HD2EXIg with human T cell type blood cellstrain Jurkat and human umbilical cord blood mononuclear CD34 positivecells was studied. In the binding experiment, human Delta-1 chimeraprotein (HD1EXIg), which was found to have the similar activity by thepresent inventors, was used as comparative experiment as like in theexample 8.

[0200] Jurkat cells, 1×10⁶ cells, were suspended in Hank's balanced saltsolution (Gibco BRL, U.S.A.) containing 2% FCS and 10 mM Hepes. HD2EXIg,HD1EXIg or human IgG1, each 1 μg/ml, were added therein and allowed tostand at 4° C. for 3 hours. Cells were washed with the Hank's solutionby centrifugation, and PE (phycoerythrin) labeled sheep anti-human IgGmonoclonal antibody 1 μg/ml was added, then the mixture was allowed tostand in ice-cooling for 30 minutes. Thereafter, the mixture was washedtwice with the Hank's solution. Analysis was performed using the flowcytometer FACScalibur (Beckton and Dekinson, U.S.A.).

[0201] Results are shown in FIG. 2. A vertical axis indicates cellcounts and a horizontal axis indicates fluorescence intensity. The upperindicates result of the control HD1EXIg and the lower indicates resultof HD2EXIg of the present invention. Staining with HD1EXIg or HD2EXIg isshown by solid line and staining with human IgG1, a control group, isshown by a broken line. In all cases, binding with Jurkat cells wasobserved. Observing the binding activities, fluorescence intensity ofHD2EXIg of the present invention was stronger than that of HD1EXIg ofthe upper. Lean fluorescence intensity of HD2EXIg was about twice-foldstronger than that of HD1EXIg. As a result, HD2EXIg is bound with Jurkatcells stronger than HD1EXIg.

[0202] Binding activity to the human umbilical cord blood mononuclearCD34 positive cells isolated by the method described in the example 7was determined by the same staining method. In this case, staining withFITC labeled anti-human CD34 antibody HPCA-2 (Beckton and DekinsonCorp., U.S.A.) was performed simultaneously in the second antibodylabeling. Data is shown only CD34 positive, i.e. FITC positive fraction.

[0203] Result is shown in FIG. 3. Result indicates that HD2EXIg andHD1EXIg similarly bind with CD34 positive cells, and binding activity ofHD2EXIg is two-fold stronger than that of HD1EXIg.

[0204] HD2EXIg stained cells were treated by cell sorter FACSvantage(Beckton and Dekinson Corp., U.S.A.) according to the attachedinstruction manual to separate cells of HD2EXIg positive fraction.

EXAMPLE 10

[0205] Effect of Cocultivation with the Novel Human Delta-2 ExpressionCells on Blood Undifferentiated Cells

[0206] FLAG chimera protein expression vector pHD2FLAG of the fulllength novel human Delta-2 prepared in the example 3 was genetransferred into mouse cell strain Balb3T3 (Physical and ChemicalInstitute, Cell Development Bank RCB0005) according to a methoddescribed in the example 4 and selection was conducted by G418 (GibcoBRL Inc., U.S.A.) according to the known method to obtain clones. Thethus obtained clones were confirmed their expression of FLAG chimeraprotein of the full length human Delta-2 according to the methoddescribed in the example 4 and clones, for which expression wasconfirmed, were used in the following experiments. The clone isdesignated as Balb/HD2FLAG.

[0207] The umbilical cord blood mononuclear CD34 positive cells obtainedby the method in the example 7 and Balb/HD2FLAG were cocultured.Cocultivation with Balb3T3, for which no gene transfer was made, wasconducted as a control.

[0208] Culture conditions are:

[0209] 1) Balb3T3 without gene transfer and no hematopoietic factor,

[0210] 2) Balb/HD2FLAG and no hematopoietic factor,

[0211] 3) Balb3T3 without gene transfer and hematopoietic factor, and

[0212] 4) Balb/HD2FLAG and hematopoietic factor.

[0213] Medium used is α-medium added with 10% FCS and 10⁻⁵M2-mercaptoethanol. In the hematopoietic factor added group, 100 ng/mlhuman SCF. 10 ng/ml human IL-3 and 100 ng/ml human IL-6 were added.Cultivation is continued for 2 weeks and medium exchange was performedthree times a week for half volume. Prior to cocultivation with humanblood cells, previously cultured Balb3T3 cells were irradiated with 250KV Peak X-ray to suppress cell growth.

[0214] Numbers of colony forming cells and LTC-IC were measured in thepre-cultivation and the experimental groups 1) to 4).

[0215] Results were as follows.

[0216] In the pre-cultivation, total cell counts were 20000 cells. Amongthose, colony forming cells were 3200 cells and LTC-IC was 220 cells.

[0217] In 1), colony forming cells and LTC-IC counts were very few forimpossible to measure.

[0218] In 2), numbers of colony forming cells were very few forimpossible to measure. LTC-IC counts were 105 cells.

[0219] In 3), numbers of colony forming cells were 26500 and LTC-ICcounts were 90.

[0220] In 4), numbers of colony forming cells were 38000 and LTC-ICcounts were 120.

[0221] Analysis of breakdown of colony forming cells in 3) and 4)indicated that in 3), only granulocyte colony was observed, however in4) erythroblast was also observed. Consequently, difference in number ofcolony forming cells were due to difference in number of erythroblastcolony.

[0222] As a result, Balb/HD2FLAG cell has colony forming action,especially erythroblast colnoy grwoth action as well as LTC-ICmaintenance action. Result indicates that using the human Delta-2expression vector of the present invention, cells having hematopoieticcell maintaining activity can be produced.

EXAMPLE 11

[0223] Preparation of Material Immobilized with the Novel Human Delta-2and its Effect

[0224] Sepharose gel immobilized with HD2EXIg prepared in the example 5was prepared. Sepharose gel used was CNBr activated Sepharose gel(Pharmacia Inc., Sweden). HD2EXIg was immobilized according to theattached instruction manual.

[0225] The thus prepared gel and the umbilical cord blood mononuclearCD34 positive cells isolated by the method in the example 7 werecultured for twenty-four hours. Cultivation was conducted in the samemedium as in the example 10. In the control group, Sepharose gelimmobilized with BSA was prepared. After cultivation, numbers of colonyforming cells were measured according to the method in the example 8. Asa result, in the gel immobilized with HD2EXIg, numbers of colony formingcells were decreased about 40%.

[0226] Consequently, the material, to which human Delta-2 of the presentinvention was immobilized, has an action against hematopoietic cells.

EXAMPLE 12

[0227] Effect of the Novel Human Delta-2 on Vascular Endothelial Cells

[0228] Fourth subculture of vascular endothelial cells, i.e. normalhuman aortic endothelial cells and normal human lung artery endothelialcells (Kurabo Co., Japan), were used. Cells were spread 5000 cells/wellin 96 well-plate for cell culture (Falcon Inc., U.S.A.) at the cultureof third subculture and were cultured in the low serum medium forendothelial cell growth (HuMedia-EG2, Kurabo Co., Japan) containinghuman recombinant EGF (Kurabo Co., Japan) 10 ng/ml and human recombinantFGF-B 5 ng/ml. At the same time, the novel human Delta-2 extracellularchimera protein (HD2EXIg) was added for final concentration of 1 μg/ml.In the comparative experiment group, same concentration of human IgG1(Athens Research and Technology Corp., U.S.A.) was added in order toobserve effect of IgGFc region. In the control group, cultivation wasconducted without adding protein except for HuMedia-EG2. Cultivation wasconducted at 37° C., under 5% carbon dioxide gas and 100% humidity for 3days, and numbers of cells were counted.

[0229] Counting for numbers of vascular endothelial cells was performedby using NR reagent set (Kurabo Co., Japan), the principle of which wasdeveloped by Borenfreund and Purerner (Journal of Tissue Culture Methods9 (1), 7-9, 1984), i.e. neutral red method, which was applied thephenomenon that vital staining pigment neutral red(3-amino-7-dimethylamino-2-methylphenazine) can only permeate throughcell membrane of living cells to accumulate in the lysosome. Absorptionat 540 nm was measured by Immunoreader (NJ-2000, Nippon Intermed Co.,Japan). As a result, in case of aortic endothelial cells, opticaldensity (OD) in the control group was 0.18±0.02, and OD in the humanIgG1 added group was almost same grade, 0.17±0.02, however OD in theHD2EXIg added group was significantly low grade, 0.11±0.01. In case ofpulmonary artery endothelial cells, optical density (OD) in the controlgroup was 0.16±0.02, and OD in the human IgG1 added group was almostsame grade, 0.16±0.02, however OD in the HD2EXIg added group wassignificantly low grade, 0.08±0.01. These results indicate that HD2EXIgsuppresses growth of vascular endothelial cells.

EXAMPLE 13

[0230] Preparation of Drug Formulation

[0231] Each polypeptide shown in the example 5, 1 mg and human serumalbumin (Midori Juji Co.) 5 mg were dissolved in distilled water 1 ml.The solution was aseptically passed through 0.22 μm filter forsterilization, dispensed into a vial and lyophilized to prepare drugformulation.

Effect of the Invention

[0232] The novel human Delta-2 molecule of the present invention can beused for effective chemicals for suppression of growth anddifferentiation of undifferentiated cells such as undifferentiated bloodcells, and can be used as pharmaceuticals and medical care materials.

1 27 1 191 PRT Homo sapiens 1 Ser Gly Val Phe Gln Leu Gln Leu Gln GluPhe Ile Asn Glu Arg Gly 1 5 10 15 Val Leu Ala Ser Gly Arg Pro Cys GluPro Gly Cys Arg Thr Phe Phe 20 25 30 Arg Val Cys Leu Lys His Phe Gln AlaVal Val Ser Pro Gly Pro Cys 35 40 45 Thr Phe Gly Thr Val Ser Thr Pro ValLeu Gly Thr Asn Ser Phe Ala 50 55 60 Val Arg Asp Asp Ser Ser Gly Gly GlyArg Asn Pro Leu Gln Leu Pro 65 70 75 80 Phe Asn Phe Thr Trp Pro Gly ThrPhe Ser Leu Ile Ile Glu Ala Trp 85 90 95 His Ala Pro Gly Asp Asp Leu ArgPro Glu Ala Leu Pro Pro Asp Ala 100 105 110 Leu Ile Ser Lys Ile Ala IleGln Gly Ser Leu Ala Val Gly Gln Asn 115 120 125 Trp Leu Leu Asp Glu GlnThr Ser Thr Leu Thr Arg Leu Arg Tyr Ser 130 135 140 Tyr Arg Val Ile CysSer Asp Asn Tyr Tyr Gly Asp Asn Cys Ser Arg 145 150 155 160 Leu Cys LysLys Arg Asn Asp His Phe Gly His Tyr Val Cys Gln Pro 165 170 175 Asp GlyAsn Leu Ser Cys Leu Pro Gly Trp Thr Gly Glu Tyr Cys 180 185 190 2 500PRT Homo sapiens 2 Ser Gly Val Phe Gln Leu Gln Leu Gln Glu Phe Ile AsnGlu Arg Gly 1 5 10 15 Val Leu Ala Ser Gly Arg Pro Cys Glu Pro Gly CysArg Thr Phe Phe 20 25 30 Arg Val Cys Leu Lys His Phe Gln Ala Val Val SerPro Gly Pro Cys 35 40 45 Thr Phe Gly Thr Val Ser Thr Pro Val Leu Gly ThrAsn Ser Phe Ala 50 55 60 Val Arg Asp Asp Ser Ser Gly Gly Gly Arg Asn ProLeu Gln Leu Pro 65 70 75 80 Phe Asn Phe Thr Trp Pro Gly Thr Phe Ser LeuIle Ile Glu Ala Trp 85 90 95 His Ala Pro Gly Asp Asp Leu Arg Pro Glu AlaLeu Pro Pro Asp Ala 100 105 110 Leu Ile Ser Lys Ile Ala Ile Gln Gly SerLeu Ala Val Gly Gln Asn 115 120 125 Trp Leu Leu Asp Glu Gln Thr Ser ThrLeu Thr Arg Leu Arg Tyr Ser 130 135 140 Tyr Arg Val Ile Cys Ser Asp AsnTyr Tyr Gly Asp Asn Cys Ser Arg 145 150 155 160 Leu Cys Lys Lys Arg AsnAsp His Phe Gly His Tyr Val Cys Gln Pro 165 170 175 Asp Gly Asn Leu SerCys Leu Pro Gly Trp Thr Gly Glu Tyr Cys Gln 180 185 190 Gln Pro Ile CysLeu Ser Gly Cys His Glu Gln Asn Gly Tyr Cys Ser 195 200 205 Lys Pro AlaGlu Cys Leu Cys Arg Pro Gly Trp Gln Gly Arg Leu Cys 210 215 220 Asn GluCys Ile Pro His Asn Gly Cys Arg His Gly Thr Cys Ser Thr 225 230 235 240Pro Trp Gln Cys Thr Cys Asp Glu Gly Trp Gly Gly Leu Phe Cys Asp 245 250255 Gln Asp Leu Asn Tyr Cys Thr His His Ser Pro Cys Lys Asn Gly Ala 260265 270 Thr Cys Ser Asn Ser Gly Gln Arg Ser Tyr Thr Cys Thr Cys Arg Pro275 280 285 Gly Tyr Thr Gly Val Asp Cys Glu Leu Glu Leu Ser Glu Cys AspSer 290 295 300 Asn Pro Cys Arg Asn Gly Gly Ser Cys Lys Asp Gln Glu AspGly Tyr 305 310 315 320 His Cys Leu Cys Pro Pro Gly Tyr Tyr Gly Leu HisCys Glu His Ser 325 330 335 Thr Leu Ser Cys Ala Asp Ser Pro Cys Phe AsnGly Gly Ser Cys Arg 340 345 350 Glu Arg Asn Gln Gly Ala Asn Tyr Ala CysGlu Cys Pro Pro Asn Phe 355 360 365 Thr Gly Ser Asn Cys Glu Lys Lys ValAsp Arg Cys Thr Ser Asn Pro 370 375 380 Cys Ala Asn Gly Gly Gln Cys LeuAsn Arg Gly Pro Ser Arg Met Cys 385 390 395 400 Arg Cys Arg Pro Gly PheThr Gly Thr Tyr Cys Glu Leu His Val Ser 405 410 415 Asp Cys Ala Arg AsnPro Cys Ala His Gly Gly Thr Cys His Asp Leu 420 425 430 Glu Asn Gly LeuMet Cys Thr Cys Pro Ala Gly Phe Ser Gly Arg Arg 435 440 445 Cys Glu ValArg Thr Ser Ile Asp Ala Cys Ala Ser Ser Pro Cys Phe 450 455 460 Asn ArgAla Thr Cys Tyr Thr Asp Leu Ser Thr Asp Thr Phe Val Cys 465 470 475 480Asn Cys Pro Tyr Gly Phe Val Gly Ser Arg Cys Glu Phe Pro Val Gly 485 490495 Leu Pro Pro Ser 500 3 659 PRT Homo sapiens 3 Ser Gly Val Phe Gln LeuGln Leu Gln Glu Phe Ile Asn Glu Arg Gly 1 5 10 15 Val Leu Ala Ser GlyArg Pro Cys Glu Pro Gly Cys Arg Thr Phe Phe 20 25 30 Arg Val Cys Leu LysHis Phe Gln Ala Val Val Ser Pro Gly Pro Cys 35 40 45 Thr Phe Gly Thr ValSer Thr Pro Val Leu Gly Thr Asn Ser Phe Ala 50 55 60 Val Arg Asp Asp SerSer Gly Gly Gly Arg Asn Pro Leu Gln Leu Pro 65 70 75 80 Phe Asn Phe ThrTrp Pro Gly Thr Phe Ser Leu Ile Ile Glu Ala Trp 85 90 95 His Ala Pro GlyAsp Asp Leu Arg Pro Glu Ala Leu Pro Pro Asp Ala 100 105 110 Leu Ile SerLys Ile Ala Ile Gln Gly Ser Leu Ala Val Gly Gln Asn 115 120 125 Trp LeuLeu Asp Glu Gln Thr Ser Thr Leu Thr Arg Leu Arg Tyr Ser 130 135 140 TyrArg Val Ile Cys Ser Asp Asn Tyr Tyr Gly Asp Asn Cys Ser Arg 145 150 155160 Leu Cys Lys Lys Arg Asn Asp His Phe Gly His Tyr Val Cys Gln Pro 165170 175 Asp Gly Asn Leu Ser Cys Leu Pro Gly Trp Thr Gly Glu Tyr Cys Gln180 185 190 Gln Pro Ile Cys Leu Ser Gly Cys His Glu Gln Asn Gly Tyr CysSer 195 200 205 Lys Pro Ala Glu Cys Leu Cys Arg Pro Gly Trp Gln Gly ArgLeu Cys 210 215 220 Asn Glu Cys Ile Pro His Asn Gly Cys Arg His Gly ThrCys Ser Thr 225 230 235 240 Pro Trp Gln Cys Thr Cys Asp Glu Gly Trp GlyGly Leu Phe Cys Asp 245 250 255 Gln Asp Leu Asn Tyr Cys Thr His His SerPro Cys Lys Asn Gly Ala 260 265 270 Thr Cys Ser Asn Ser Gly Gln Arg SerTyr Thr Cys Thr Cys Arg Pro 275 280 285 Gly Tyr Thr Gly Val Asp Cys GluLeu Glu Leu Ser Glu Cys Asp Ser 290 295 300 Asn Pro Cys Arg Asn Gly GlySer Cys Lys Asp Gln Glu Asp Gly Tyr 305 310 315 320 His Cys Leu Cys ProPro Gly Tyr Tyr Gly Leu His Cys Glu His Ser 325 330 335 Thr Leu Ser CysAla Asp Ser Pro Cys Phe Asn Gly Gly Ser Cys Arg 340 345 350 Glu Arg AsnGln Gly Ala Asn Tyr Ala Cys Glu Cys Pro Pro Asn Phe 355 360 365 Thr GlySer Asn Cys Glu Lys Lys Val Asp Arg Cys Thr Ser Asn Pro 370 375 380 CysAla Asn Gly Gly Gln Cys Leu Asn Arg Gly Pro Ser Arg Met Cys 385 390 395400 Arg Cys Arg Pro Gly Phe Thr Gly Thr Tyr Cys Glu Leu His Val Ser 405410 415 Asp Cys Ala Arg Asn Pro Cys Ala His Gly Gly Thr Cys His Asp Leu420 425 430 Glu Asn Gly Leu Met Cys Thr Cys Pro Ala Gly Phe Ser Gly ArgArg 435 440 445 Cys Glu Val Arg Thr Ser Ile Asp Ala Cys Ala Ser Ser ProCys Phe 450 455 460 Asn Arg Ala Thr Cys Tyr Thr Asp Leu Ser Thr Asp ThrPhe Val Cys 465 470 475 480 Asn Cys Pro Tyr Gly Phe Val Gly Ser Arg CysGlu Phe Pro Val Gly 485 490 495 Leu Pro Pro Ser Phe Pro Trp Val Ala ValSer Leu Gly Val Gly Leu 500 505 510 Ala Val Leu Leu Val Leu Leu Gly MetVal Ala Val Ala Val Arg Gln 515 520 525 Leu Arg Leu Arg Arg Pro Asp AspGly Ser Arg Glu Ala Met Asn Asn 530 535 540 Leu Ser Asp Phe Gln Lys AspAsn Leu Ile Pro Ala Ala Gln Leu Lys 545 550 555 560 Asn Thr Asn Gln LysLys Glu Leu Glu Val Asp Cys Gly Leu Asp Lys 565 570 575 Ser Asn Cys GlyLys Gln Gln Asn His Thr Leu Asp Tyr Asn Leu Ala 580 585 590 Pro Gly ProLeu Gly Arg Gly Thr Met Pro Gly Lys Phe Pro His Ser 595 600 605 Asp LysSer Leu Gly Glu Lys Ala Pro Leu Arg Leu His Ser Glu Lys 610 615 620 ProGlu Cys Arg Ile Ser Ala Ile Cys Ser Pro Arg Asp Ser Met Tyr 625 630 635640 Gln Ser Val Cys Leu Ile Ser Glu Glu Arg Asn Glu Cys Val Ile Ala 645650 655 Thr Glu Val 4 3339 DNA Homo sapiens CDS (277)..(2331)sig_peptide (277)..(354) mat_peptide (355)..(2331) 4 gccgccttggtgcagcgtac accggcacta gcccgcttgc agccccagga ttagacagaa 60 gacgcgtcctcggcgcggtc gccgcccagc cgtagtcacc tggattacct acagcggcag 120 ctgcagcggagccagcgaga aggccaaagg ggagcagcgt cccgagagga gcgcctcttt 180 tcagggaccccgccggctgg cggacgcgcg ggaaagcggc gtcgcgaaca gagccagatt 240 gagggcccgcgggtggagag agcgacgccc gagggg atg gcg gca gcg tcc cgg 294 Met Ala Ala AlaSer Arg -25 agc gcc tct ggc tgg gcg cta ctg ctg ctg gtg gca ctt tgg cagcag 342 Ser Ala Ser Gly Trp Ala Leu Leu Leu Leu Val Ala Leu Trp Gln Gln-20 -15 -10 -5 cgc gcg gcc ggc tcc ggc gtc ttc cag ctg cag ctg cag gagttc atc 390 Arg Ala Ala Gly Ser Gly Val Phe Gln Leu Gln Leu Gln Glu PheIle -1 1 5 10 aac gag cgc ggc gta ctg gcc agt ggg cgg cct tgc gag cccggc tgc 438 Asn Glu Arg Gly Val Leu Ala Ser Gly Arg Pro Cys Glu Pro GlyCys 15 20 25 cgg act ttc ttc cgc gtc tgc ctt aag cac ttc cag gcg gtc gtctcg 486 Arg Thr Phe Phe Arg Val Cys Leu Lys His Phe Gln Ala Val Val Ser30 35 40 ccc gga ccc tgc acc ttc ggg acc gtc tcc acg ccg gta ttg ggc acc534 Pro Gly Pro Cys Thr Phe Gly Thr Val Ser Thr Pro Val Leu Gly Thr 4550 55 60 aac tcc ttc gct gtc cgg gac gac agt agc ggc ggg ggg cgc aac cct582 Asn Ser Phe Ala Val Arg Asp Asp Ser Ser Gly Gly Gly Arg Asn Pro 6570 75 ctc caa ctg ccc ttc aat ttc acc tgg ccg ggt acc ttc tcg ctc atc630 Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro Gly Thr Phe Ser Leu Ile 8085 90 atc gaa gct tgg cac gcg cca gga gac gac ctg cgg cca gag gcc ttg678 Ile Glu Ala Trp His Ala Pro Gly Asp Asp Leu Arg Pro Glu Ala Leu 95100 105 cca cca gat gca ctc atc agc aag atc gcc atc cag ggc tcc cta gct726 Pro Pro Asp Ala Leu Ile Ser Lys Ile Ala Ile Gln Gly Ser Leu Ala 110115 120 gtg ggt cag aac tgg tta ttg gat gag caa acc agc acc ctc aca agg774 Val Gly Gln Asn Trp Leu Leu Asp Glu Gln Thr Ser Thr Leu Thr Arg 125130 135 140 ctg cgc tac tct tac cgg gtc atc tgc agt gac aac tac tat ggagac 822 Leu Arg Tyr Ser Tyr Arg Val Ile Cys Ser Asp Asn Tyr Tyr Gly Asp145 150 155 aac tgc tcc cgc ctg tgc aag aag cgc aat gac cac ttc ggc cactat 870 Asn Cys Ser Arg Leu Cys Lys Lys Arg Asn Asp His Phe Gly His Tyr160 165 170 gtg tgc cag cca gat ggc aac ttg tcc tgc ctg ccc ggt tgg actggg 918 Val Cys Gln Pro Asp Gly Asn Leu Ser Cys Leu Pro Gly Trp Thr Gly175 180 185 gaa tat tgc caa cag cct atc tgt ctt tcg ggc tgt cat gaa cagaat 966 Glu Tyr Cys Gln Gln Pro Ile Cys Leu Ser Gly Cys His Glu Gln Asn190 195 200 ggc tac tgc agc aag cca gca gag tgc ctc tgc cgc cca ggc tggcag 1014 Gly Tyr Cys Ser Lys Pro Ala Glu Cys Leu Cys Arg Pro Gly Trp Gln205 210 215 220 ggc cgg ctg tgt aac gaa tgc atc ccc cac aat ggc tgt cgccac ggc 1062 Gly Arg Leu Cys Asn Glu Cys Ile Pro His Asn Gly Cys Arg HisGly 225 230 235 acc tgc agc act ccc tgg caa tgt act tgt gat gag ggc tgggga ggc 1110 Thr Cys Ser Thr Pro Trp Gln Cys Thr Cys Asp Glu Gly Trp GlyGly 240 245 250 ctg ttt tgt gac caa gat ctc aac tac tgc acc cac cac tcccca tgc 1158 Leu Phe Cys Asp Gln Asp Leu Asn Tyr Cys Thr His His Ser ProCys 255 260 265 aag aat ggg gca acg tgc tcc aac agt ggg cag cga agc tacacc tgc 1206 Lys Asn Gly Ala Thr Cys Ser Asn Ser Gly Gln Arg Ser Tyr ThrCys 270 275 280 acc tgt cgc cca ggc tac act ggt gtg gac tgt gag ctg gagctc agc 1254 Thr Cys Arg Pro Gly Tyr Thr Gly Val Asp Cys Glu Leu Glu LeuSer 285 290 295 300 gag tgt gac agc aac ccc tgt cgc aat gga ggc agc tgtaag gac cag 1302 Glu Cys Asp Ser Asn Pro Cys Arg Asn Gly Gly Ser Cys LysAsp Gln 305 310 315 gag gat ggc tac cac tgc ctg tgt cct ccg ggc tac tatggc ctg cat 1350 Glu Asp Gly Tyr His Cys Leu Cys Pro Pro Gly Tyr Tyr GlyLeu His 320 325 330 tgt gaa cac agc acc ttg agc tgc gcc gac tcc ccc tgcttc aat ggg 1398 Cys Glu His Ser Thr Leu Ser Cys Ala Asp Ser Pro Cys PheAsn Gly 335 340 345 ggc tcc tgc cgg gag cgc aac cag ggg gcc aac tat gcttgt gaa tgt 1446 Gly Ser Cys Arg Glu Arg Asn Gln Gly Ala Asn Tyr Ala CysGlu Cys 350 355 360 ccc ccc aac ttc acc ggc tcc aac tgc gag aag aaa gtggac agg tgc 1494 Pro Pro Asn Phe Thr Gly Ser Asn Cys Glu Lys Lys Val AspArg Cys 365 370 375 380 acc agc aac ccc tgt gcc aac ggg gga cag tgc ctgaac cga ggt cca 1542 Thr Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu AsnArg Gly Pro 385 390 395 agc cgc atg tgc cgc tgc cgt cct gga ttc acg ggcacc tac tgt gaa 1590 Ser Arg Met Cys Arg Cys Arg Pro Gly Phe Thr Gly ThrTyr Cys Glu 400 405 410 ctc cac gtc agc gac tgt gcc cgt aac cct tgc gcccac ggt ggc act 1638 Leu His Val Ser Asp Cys Ala Arg Asn Pro Cys Ala HisGly Gly Thr 415 420 425 tgc cat gac ctg gag aat ggg ctc atg tgc acc tgccct gcc ggc ttc 1686 Cys His Asp Leu Glu Asn Gly Leu Met Cys Thr Cys ProAla Gly Phe 430 435 440 tct ggc cga cgc tgt gag gtg cgg aca tcc atc gatgcc tgt gcc tcg 1734 Ser Gly Arg Arg Cys Glu Val Arg Thr Ser Ile Asp AlaCys Ala Ser 445 450 455 460 agt ccc tgc ttc aac agg gcc acc tgc tac accgac ctc tcc aca gac 1782 Ser Pro Cys Phe Asn Arg Ala Thr Cys Tyr Thr AspLeu Ser Thr Asp 465 470 475 acc ttt gtg tgc aac tgc cct tat ggc ttt gtgggc agc cgc tgc gag 1830 Thr Phe Val Cys Asn Cys Pro Tyr Gly Phe Val GlySer Arg Cys Glu 480 485 490 ttc ccc gtg ggc ttg ccg ccc agc ttc ccc tgggtg gcc gtc tcg ctg 1878 Phe Pro Val Gly Leu Pro Pro Ser Phe Pro Trp ValAla Val Ser Leu 495 500 505 ggt gtg ggg ctg gca gtg ctg ctg gta ctg ctgggc atg gtg gca gtg 1926 Gly Val Gly Leu Ala Val Leu Leu Val Leu Leu GlyMet Val Ala Val 510 515 520 gct gtg cgg cag ctg cgg ctt cga cgg ccg gacgac ggc agc agg gaa 1974 Ala Val Arg Gln Leu Arg Leu Arg Arg Pro Asp AspGly Ser Arg Glu 525 530 535 540 gcc atg aac aac ttg tcg gac ttc cag aaggac aac ctg att cct gcc 2022 Ala Met Asn Asn Leu Ser Asp Phe Gln Lys AspAsn Leu Ile Pro Ala 545 550 555 gcc cag ctt aaa aac aca aac cag aag aaggag ctg gaa gtg gac tgt 2070 Ala Gln Leu Lys Asn Thr Asn Gln Lys Lys GluLeu Glu Val Asp Cys 560 565 570 ggc ctg gac aag tcc aac tgt ggc aaa cagcaa aac cac aca ttg gac 2118 Gly Leu Asp Lys Ser Asn Cys Gly Lys Gln GlnAsn His Thr Leu Asp 575 580 585 tat aat ctg gcc cca ggg ccc ctg ggg cggggg acc atg cca gga aag 2166 Tyr Asn Leu Ala Pro Gly Pro Leu Gly Arg GlyThr Met Pro Gly Lys 590 595 600 ttt ccc cac agt gac aag agc tta gga gagaag gcg cca ctg cgg tta 2214 Phe Pro His Ser Asp Lys Ser Leu Gly Glu LysAla Pro Leu Arg Leu 605 610 615 620 cac agt gaa aag cca gag tgt cgg atatca gcg ata tgc tcc ccc agg 2262 His Ser Glu Lys Pro Glu Cys Arg Ile SerAla Ile Cys Ser Pro Arg 625 630 635 gac tcc atg tac cag tct gtg tgt ttgata tca gag gag agg aat gaa 2310 Asp Ser Met Tyr Gln Ser Val Cys Leu IleSer Glu Glu Arg Asn Glu 640 645 650 tgt gtc att gcc acg gag gtataaggcagga gcctacctgg acatccctgc 2361 Cys Val Ile Ala Thr Glu Val 655tcagccccgc ggctggacct tccttctgca ttgtttacat tgcatcctgg atgggacgtt 2421tttcatatgc aacgtgctgc tctcaggagg aggagggaat ggcaggaacc ggacagactg 2481tgaacttgcc aagagatgca atacccttcc acacctttgg gtgtctgtct ggcatcagat 2541tggcagctgc accaaccaga ggaacagaag agaagagaga tgccactggg cactgccctg 2601ccagtagtgg ccttcagggg gctccttccg gggctccggc ctgttttcca gagagagtgg 2661cagtagcccc atggggcccg gagctgctgt ggcctccact ggcatccgtg tttccaaaag 2721tgcctttggc ccaggctcca cggcgacagt tgggcccaaa tcagaaagga gagagggggc 2781caatgagggc agggcctcct gtgggctgga aaaccactgg gtgcgtctct tgctggggtt 2841tgccctggag gtgaggtgag tgctcgaggg aggggagtgc tttctgcccc atgcctccaa 2901ctactgtatg caggcctggc tctctggtct aggccctttg ggcaagaatg tccgtctacc 2961cggcttccac caccctctgg ccctgggctt ctgtaagcag acaggcagag ggcctgcccc 3021tcccaccagc caagggtgcc aggcctaact ggggcactca gggcagtgtg ttggaaattc 3081cactgagggg gaaatcaggt gctgcggccg cctgggccct ttcctccctc aagcccatct 3141ccacaacctc gagcctgggc tctggtccac tactgcccca gaccaccctc aaagctggtc 3201ttcagaaatc aataatatga gtttttattt tgtttttttt tttttttttg tagtttattt 3261tggagtctag tatttcaata atttaagaat cagaagcact gacctttcta cattttataa 3321cattattttg tatataat 3339 5 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 5 gat tat aaa gat gat gat gat aaa tga27 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 6 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 6 tggcartgya aytgycarga20 7 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 7 atyttyttyt crcarttraa 20 8 2663 DNA Homo sapiens CDS(179)..(2347) sig_peptide (179)..(241) mat_peptide (242)..(2347) 8cttgggaaga ggcggagacc ggcttttaaa gaaagaagtc ctgggtcctg cggtctgggg 60cgaggcaagg gcgcttttct gcccacgctc cccgtggccc atcgatcccc cgcgcgtccg 120ccgctgttct aaggagagaa gtgggggccc cccaggctcg cgcgtggagc gaagcagc 178 atgggc agt cgg tgc gcg ctg gcc ctg gcg gtg ctc tcg gcc ttg ctg 226 Met GlySer Arg Cys Ala Leu Ala Leu Ala Val Leu Ser Ala Leu Leu -20 -15 -10 tgtcag gtc tgg agc tct ggg gtg ttc gaa ctg aag ctg cag gag ttc 274 Cys GlnVal Trp Ser Ser Gly Val Phe Glu Leu Lys Leu Gln Glu Phe -5 -1 1 5 10 gtcaac aag aag ggg ctg ctg ggg aac cgc aac tgc tgc cgc ggg ggc 322 Val AsnLys Lys Gly Leu Leu Gly Asn Arg Asn Cys Cys Arg Gly Gly 15 20 25 gcg gggcca ccg ccg tgc gcc tgc cgg acc ttc ttc cgc gtg tgc ctc 370 Ala Gly ProPro Pro Cys Ala Cys Arg Thr Phe Phe Arg Val Cys Leu 30 35 40 aag cac taccag gcc agc gtg tcc ccc gag ccg ccc tgc acc tac ggc 418 Lys His Tyr GlnAla Ser Val Ser Pro Glu Pro Pro Cys Thr Tyr Gly 45 50 55 agc gcc gtc accccc gtg ctg ggc gtc gac tcc ttc agt ctg ccc gac 466 Ser Ala Val Thr ProVal Leu Gly Val Asp Ser Phe Ser Leu Pro Asp 60 65 70 75 ggc ggg ggc gccgac tcc gcg ttc agc aac ccc atc cgc ttc ccc ttc 514 Gly Gly Gly Ala AspSer Ala Phe Ser Asn Pro Ile Arg Phe Pro Phe 80 85 90 ggc ttc acc tgg ccgggc acc ttc tct ctg att att gaa gct ctc cac 562 Gly Phe Thr Trp Pro GlyThr Phe Ser Leu Ile Ile Glu Ala Leu His 95 100 105 aca gat tct cct gatgac ctc gca aca gaa aac cca gaa aga ctc atc 610 Thr Asp Ser Pro Asp AspLeu Ala Thr Glu Asn Pro Glu Arg Leu Ile 110 115 120 agc cgc ctg gcc acccag agg cac ctg acg gtg ggc gag gag tgg tcc 658 Ser Arg Leu Ala Thr GlnArg His Leu Thr Val Gly Glu Glu Trp Ser 125 130 135 cag gac ctg cac agcagc ggc cgc acg gac ctc aag tac tcc tac cgc 706 Gln Asp Leu His Ser SerGly Arg Thr Asp Leu Lys Tyr Ser Tyr Arg 140 145 150 155 ttc gtg tgt gacgaa cac tac tac gga gag ggc tgc tcc gtt ttc tgc 754 Phe Val Cys Asp GluHis Tyr Tyr Gly Glu Gly Cys Ser Val Phe Cys 160 165 170 cgt ccc cgg gacgat gcc ttc ggc cac ttc acc tgt ggg gag cgt ggg 802 Arg Pro Arg Asp AspAla Phe Gly His Phe Thr Cys Gly Glu Arg Gly 175 180 185 gag aaa gtg tgcaac cct ggc tgg aaa ggg ccc tac tgc aca gag ccg 850 Glu Lys Val Cys AsnPro Gly Trp Lys Gly Pro Tyr Cys Thr Glu Pro 190 195 200 atc tgc ctg cctgga tgt gat gag cag cat gga ttt tgt gac aaa cca 898 Ile Cys Leu Pro GlyCys Asp Glu Gln His Gly Phe Cys Asp Lys Pro 205 210 215 ggg gaa tgc aagtgc aga gtg ggc tgg cag ggc cgg tac tgt gac gag 946 Gly Glu Cys Lys CysArg Val Gly Trp Gln Gly Arg Tyr Cys Asp Glu 220 225 230 235 tgt atc cgctat cca ggc tgt ctc cat ggc acc tgc cag cag ccc tgg 994 Cys Ile Arg TyrPro Gly Cys Leu His Gly Thr Cys Gln Gln Pro Trp 240 245 250 cag tgc aactgc cag gaa ggc tgg ggg ggc ctt ttc tgc aac cag gac 1042 Gln Cys Asn CysGln Glu Gly Trp Gly Gly Leu Phe Cys Asn Gln Asp 255 260 265 ctg aac tactgc aca cac cat aag ccc tgc aag aat gga gcc acc tgc 1090 Leu Asn Tyr CysThr His His Lys Pro Cys Lys Asn Gly Ala Thr Cys 270 275 280 acc aac acgggc cag ggg agc tac act tgc tct tgc cgg cct ggg tac 1138 Thr Asn Thr GlyGln Gly Ser Tyr Thr Cys Ser Cys Arg Pro Gly Tyr 285 290 295 aca ggt gccacc tgc gag ctg ggg att gac gag tgt gac ccc agc cct 1186 Thr Gly Ala ThrCys Glu Leu Gly Ile Asp Glu Cys Asp Pro Ser Pro 300 305 310 315 tgt aagaac gga ggg agc tgc acg gat ctc gag aac agc tac tcc tgt 1234 Cys Lys AsnGly Gly Ser Cys Thr Asp Leu Glu Asn Ser Tyr Ser Cys 320 325 330 acc tgccca ccc ggc ttc tac ggc aaa atc tgt gaa ttg agt gcc atg 1282 Thr Cys ProPro Gly Phe Tyr Gly Lys Ile Cys Glu Leu Ser Ala Met 335 340 345 acc tgtgcg gac ggc cct tgc ttt aac ggg ggt cgg tgc tca gac agc 1330 Thr Cys AlaAsp Gly Pro Cys Phe Asn Gly Gly Arg Cys Ser Asp Ser 350 355 360 ccc gatgga ggg tac agc tgc cgc tgc ccc gtg ggc tac tcc ggc ttc 1378 Pro Asp GlyGly Tyr Ser Cys Arg Cys Pro Val Gly Tyr Ser Gly Phe 365 370 375 aac tgtgag aag aaa att gac tac tgc agc tct tca ccc tgt tct aat 1426 Asn Cys GluLys Lys Ile Asp Tyr Cys Ser Ser Ser Pro Cys Ser Asn 380 385 390 395 ggtgcc aag tgt gtg gac ctc ggt gat gcc tac ctg tgc cgc tgc cag 1474 Gly AlaLys Cys Val Asp Leu Gly Asp Ala Tyr Leu Cys Arg Cys Gln 400 405 410 gccggc ttc tcg ggg agg cac tgt gac gac aac gtg gac gac tgc gcc 1522 Ala GlyPhe Ser Gly Arg His Cys Asp Asp Asn Val Asp Asp Cys Ala 415 420 425 tcctcc ccg tgc gcc aac ggg ggc acc tgc cgg gat ggc gtg aac gac 1570 Ser SerPro Cys Ala Asn Gly Gly Thr Cys Arg Asp Gly Val Asn Asp 430 435 440 ttctcc tgc acc tgc ccg cct ggc tac acg ggc agg aac tgc agt gcc 1618 Phe SerCys Thr Cys Pro Pro Gly Tyr Thr Gly Arg Asn Cys Ser Ala 445 450 455 cccgtc agc agg tgc gag cac gca ccc tgc cac aat ggg gcc acc tgc 1666 Pro ValSer Arg Cys Glu His Ala Pro Cys His Asn Gly Ala Thr Cys 460 465 470 475cac gag agg ggc cac cgc tat gtg tgc gag tgt gcc cga ggc tac ggg 1714 HisGlu Arg Gly His Arg Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly 480 485 490ggt ccc aac tgc cag ttc ctg ctc ccc gag ctg ccc ccg ggc cca gcg 1762 GlyPro Asn Cys Gln Phe Leu Leu Pro Glu Leu Pro Pro Gly Pro Ala 495 500 505gtg gtg gac ctc act gag aag cta gag ggc cag ggc ggg cca ttc ccc 1810 ValVal Asp Leu Thr Glu Lys Leu Glu Gly Gln Gly Gly Pro Phe Pro 510 515 520tgg gtg gcc gtg tgc gcc ggg gtc atc ctt gtc ctc atg ctg ctg ctg 1858 TrpVal Ala Val Cys Ala Gly Val Ile Leu Val Leu Met Leu Leu Leu 525 530 535ggc tgt gcc gct gtg gtg gtc tgc gtc cgg ctg agg ctg cag aag cac 1906 GlyCys Ala Ala Val Val Val Cys Val Arg Leu Arg Leu Gln Lys His 540 545 550555 cgg ccc cca gcc gac ccc tgc cgg ggg gag acg gag acc atg aac aac 1954Arg Pro Pro Ala Asp Pro Cys Arg Gly Glu Thr Glu Thr Met Asn Asn 560 565570 ctg gcc aac tgc cag cgt gag aag gac atc tca gtc agc atc atc ggg 2002Leu Ala Asn Cys Gln Arg Glu Lys Asp Ile Ser Val Ser Ile Ile Gly 575 580585 gcc acg cag atc aag aac acc aac aag aag gcg gac ttc cac ggg gac 2050Ala Thr Gln Ile Lys Asn Thr Asn Lys Lys Ala Asp Phe His Gly Asp 590 595600 cac agc gcc gac aag aat ggc ttc aag gcc cgc tac cca gcg gtg gac 2098His Ser Ala Asp Lys Asn Gly Phe Lys Ala Arg Tyr Pro Ala Val Asp 605 610615 tat aac ctc gtg cag gac ctc aag ggt gac gac acc gcc gtc agg gac 2146Tyr Asn Leu Val Gln Asp Leu Lys Gly Asp Asp Thr Ala Val Arg Asp 620 625630 635 gcg cac agc aag cgt gac acc aag tgc cag ccc cag ggc tcc tca ggg2194 Ala His Ser Lys Arg Asp Thr Lys Cys Gln Pro Gln Gly Ser Ser Gly 640645 650 gag gag aag ggg acc ccg acc aca ctc agg ggt gga gaa gca tct gaa2242 Glu Glu Lys Gly Thr Pro Thr Thr Leu Arg Gly Gly Glu Ala Ser Glu 655660 665 aga aaa agg ccg gac tcg ggc tgt tca act tca aaa gac acc aag tac2290 Arg Lys Arg Pro Asp Ser Gly Cys Ser Thr Ser Lys Asp Thr Lys Tyr 670675 680 cag tcg gtg tac gtc ata tcc gag gag aag gat gag tgc gtc ata gca2338 Gln Ser Val Tyr Val Ile Ser Glu Glu Lys Asp Glu Cys Val Ile Ala 685690 695 act gag gtg taaaatggaa gtgagatggc aagactcccg tttctcttaa 2387 ThrGlu Val 700 aataagtaaa attccaagga tatatgcccc aacgaatgct gctgaagaggagggaggcct 2447 cgtggactgc tgctgagaaa ccgagttcag accgagcagg ttctcctcctgaggtcctcg 2507 acgcctgccg acagcctgtc gcggcccggc cgcctgcggc actgccttccgtgacgtcgc 2567 cgttgcacta tggacagttg ctcttaagag aatatatatt taaatgggtgaactgaatta 2627 cgcataagaa gcatgcactg cctgagtgta tatttt 2663 9 20 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 9tgacggtggg cgaggagtgg 20 10 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 10 gcagctgtac cctccatcgg 20 11 716 DNAHomo sapiens 11 tgacggtggg cgaggagtgg tcccaggacc tgcacagcag cggccgcacggacctcaagt 60 actcctaccg cttcgtgtgt gacgaacact actacggaga gggctgctccgttttctgcc 120 gtccccggga cgatgccttc ggccacttca cctgtgggga gcgtggggagaaagtgtgca 180 accctggctg gaaagggccc tactgcacag agccgatctg cctgcctggatgtgatgagc 240 agcatggatt ttgtgacaaa ccaggggaat gcaagtgcag agtgggctggcagggccggt 300 actgtgacga gtgtatccgc tatccaggct gtctccatgg cacctgccagcagccctggc 360 agtgcaactg ccaggaaggc tgggggggcc ttttctgcaa ccaggacctgaactactgca 420 cacaccataa gccctgcaag aatggagcca cctgcaccaa cacgggccaggggagctaca 480 cttgctcttg ccggcctggg tacacaggtg ccacctgcga gctggggattgacgagtgtg 540 accccagccc ttgtaagaac ggagggagct gcacggatct cgagaacagctactcctgta 600 cctgcccacc cggcttctac ggcaaaatct gtgaattgag tgccatgacctgtgcggacg 660 gcccttgctt taacgggggt cggtgctcag acagccccga tggagggtacagctgc 716 12 20 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 12 ttgggcacca actccttcgc 20 13 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic DNA 13 taggctgttggcaatattcc 20 14 412 DNA Homo sapiens 14 ttgggcacca actccttcgctgtccgggac gacagtagcg gcggggggcg caaccctctc 60 caactgccct tcaatttcacctggccgggt accttctcgc tcatcatcga agcttggcac 120 gcgccaggag acgacctgcggccagaggcc ttgccaccag atgcactcat cagcaagatc 180 gccatccagg gctccctagctgtgggtcag aactggttat tggatgagca aaccagcacc 240 ctcacaaggc tgcgctactcttaccgggtc atctgcagtg acaactacta tggagacaac 300 tgctcccgcc tgtgcaagaagcgcaatgac cacttcggcc actatgtgtg ccagccagat 360 ggcaacttgt cctgcctgcccggttggact ggggaatatt gccaacagcc ta 412 15 28 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 15 cggcgacgcccgaggggatg gcggcagc 28 16 30 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 16 gaattccacc gcggtggagc tccaattcgc 3017 28 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 17 tcagctgggc ggcaagccca cggggaac 28 18 33 DNA ArtificialSequence Description of Artificial Sequence Synthetic DNA 18 gcggccgcttatcgataccg tcgacctcga ggg 33 19 52 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic DNA 19 tcatttatca tcatcatctt tataatcgctgggcggcaag cccacgggga ac 52 20 36 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 20 aaaggatccg agggcggcaa gcccacggggaactcg 36 21 25 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 21 ttatacctcc gtggcaatga cacat 25 22 52 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 22tcatttatca tcatcatctt tataatctac ctccgtggca atgacacatt ca 52 23 36 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 23aaggatcccg agggtgtctg ctggaagcca ggctca 36 24 33 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 24 cctctagagtcgcggccgtc gcactcattt acc 33 25 685 PRT Homo sapiens 25 Met Ala Ala AlaSer Arg Ser Ala Ser Gly Trp Ala Leu Leu Leu Leu -25 -20 -15 Val Ala LeuTrp Gln Gln Arg Ala Ala Gly Ser Gly Val Phe Gln Leu -10 -5 -1 1 5 GlnLeu Gln Glu Phe Ile Asn Glu Arg Gly Val Leu Ala Ser Gly Arg 10 15 20 ProCys Glu Pro Gly Cys Arg Thr Phe Phe Arg Val Cys Leu Lys His 25 30 35 PheGln Ala Val Val Ser Pro Gly Pro Cys Thr Phe Gly Thr Val Ser 40 45 50 ThrPro Val Leu Gly Thr Asn Ser Phe Ala Val Arg Asp Asp Ser Ser 55 60 65 70Gly Gly Gly Arg Asn Pro Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro 75 80 85Gly Thr Phe Ser Leu Ile Ile Glu Ala Trp His Ala Pro Gly Asp Asp 90 95100 Leu Arg Pro Glu Ala Leu Pro Pro Asp Ala Leu Ile Ser Lys Ile Ala 105110 115 Ile Gln Gly Ser Leu Ala Val Gly Gln Asn Trp Leu Leu Asp Glu Gln120 125 130 Thr Ser Thr Leu Thr Arg Leu Arg Tyr Ser Tyr Arg Val Ile CysSer 135 140 145 150 Asp Asn Tyr Tyr Gly Asp Asn Cys Ser Arg Leu Cys LysLys Arg Asn 155 160 165 Asp His Phe Gly His Tyr Val Cys Gln Pro Asp GlyAsn Leu Ser Cys 170 175 180 Leu Pro Gly Trp Thr Gly Glu Tyr Cys Gln GlnPro Ile Cys Leu Ser 185 190 195 Gly Cys His Glu Gln Asn Gly Tyr Cys SerLys Pro Ala Glu Cys Leu 200 205 210 Cys Arg Pro Gly Trp Gln Gly Arg LeuCys Asn Glu Cys Ile Pro His 215 220 225 230 Asn Gly Cys Arg His Gly ThrCys Ser Thr Pro Trp Gln Cys Thr Cys 235 240 245 Asp Glu Gly Trp Gly GlyLeu Phe Cys Asp Gln Asp Leu Asn Tyr Cys 250 255 260 Thr His His Ser ProCys Lys Asn Gly Ala Thr Cys Ser Asn Ser Gly 265 270 275 Gln Arg Ser TyrThr Cys Thr Cys Arg Pro Gly Tyr Thr Gly Val Asp 280 285 290 Cys Glu LeuGlu Leu Ser Glu Cys Asp Ser Asn Pro Cys Arg Asn Gly 295 300 305 310 GlySer Cys Lys Asp Gln Glu Asp Gly Tyr His Cys Leu Cys Pro Pro 315 320 325Gly Tyr Tyr Gly Leu His Cys Glu His Ser Thr Leu Ser Cys Ala Asp 330 335340 Ser Pro Cys Phe Asn Gly Gly Ser Cys Arg Glu Arg Asn Gln Gly Ala 345350 355 Asn Tyr Ala Cys Glu Cys Pro Pro Asn Phe Thr Gly Ser Asn Cys Glu360 365 370 Lys Lys Val Asp Arg Cys Thr Ser Asn Pro Cys Ala Asn Gly GlyGln 375 380 385 390 Cys Leu Asn Arg Gly Pro Ser Arg Met Cys Arg Cys ArgPro Gly Phe 395 400 405 Thr Gly Thr Tyr Cys Glu Leu His Val Ser Asp CysAla Arg Asn Pro 410 415 420 Cys Ala His Gly Gly Thr Cys His Asp Leu GluAsn Gly Leu Met Cys 425 430 435 Thr Cys Pro Ala Gly Phe Ser Gly Arg ArgCys Glu Val Arg Thr Ser 440 445 450 Ile Asp Ala Cys Ala Ser Ser Pro CysPhe Asn Arg Ala Thr Cys Tyr 455 460 465 470 Thr Asp Leu Ser Thr Asp ThrPhe Val Cys Asn Cys Pro Tyr Gly Phe 475 480 485 Val Gly Ser Arg Cys GluPhe Pro Val Gly Leu Pro Pro Ser Phe Pro 490 495 500 Trp Val Ala Val SerLeu Gly Val Gly Leu Ala Val Leu Leu Val Leu 505 510 515 Leu Gly Met ValAla Val Ala Val Arg Gln Leu Arg Leu Arg Arg Pro 520 525 530 Asp Asp GlySer Arg Glu Ala Met Asn Asn Leu Ser Asp Phe Gln Lys 535 540 545 550 AspAsn Leu Ile Pro Ala Ala Gln Leu Lys Asn Thr Asn Gln Lys Lys 555 560 565Glu Leu Glu Val Asp Cys Gly Leu Asp Lys Ser Asn Cys Gly Lys Gln 570 575580 Gln Asn His Thr Leu Asp Tyr Asn Leu Ala Pro Gly Pro Leu Gly Arg 585590 595 Gly Thr Met Pro Gly Lys Phe Pro His Ser Asp Lys Ser Leu Gly Glu600 605 610 Lys Ala Pro Leu Arg Leu His Ser Glu Lys Pro Glu Cys Arg IleSer 615 620 625 630 Ala Ile Cys Ser Pro Arg Asp Ser Met Tyr Gln Ser ValCys Leu Ile 635 640 645 Ser Glu Glu Arg Asn Glu Cys Val Ile Ala Thr GluVal 650 655 26 8 PRT Artificial Sequence Description of ArtificialSequence Synthetic amino acid 26 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 27723 PRT Homo sapiens 27 Met Gly Ser Arg Cys Ala Leu Ala Leu Ala Val LeuSer Ala Leu Leu -20 -15 -10 Cys Gln Val Trp Ser Ser Gly Val Phe Glu LeuLys Leu Gln Glu Phe -5 -1 1 5 10 Val Asn Lys Lys Gly Leu Leu Gly Asn ArgAsn Cys Cys Arg Gly Gly 15 20 25 Ala Gly Pro Pro Pro Cys Ala Cys Arg ThrPhe Phe Arg Val Cys Leu 30 35 40 Lys His Tyr Gln Ala Ser Val Ser Pro GluPro Pro Cys Thr Tyr Gly 45 50 55 Ser Ala Val Thr Pro Val Leu Gly Val AspSer Phe Ser Leu Pro Asp 60 65 70 75 Gly Gly Gly Ala Asp Ser Ala Phe SerAsn Pro Ile Arg Phe Pro Phe 80 85 90 Gly Phe Thr Trp Pro Gly Thr Phe SerLeu Ile Ile Glu Ala Leu His 95 100 105 Thr Asp Ser Pro Asp Asp Leu AlaThr Glu Asn Pro Glu Arg Leu Ile 110 115 120 Ser Arg Leu Ala Thr Gln ArgHis Leu Thr Val Gly Glu Glu Trp Ser 125 130 135 Gln Asp Leu His Ser SerGly Arg Thr Asp Leu Lys Tyr Ser Tyr Arg 140 145 150 155 Phe Val Cys AspGlu His Tyr Tyr Gly Glu Gly Cys Ser Val Phe Cys 160 165 170 Arg Pro ArgAsp Asp Ala Phe Gly His Phe Thr Cys Gly Glu Arg Gly 175 180 185 Glu LysVal Cys Asn Pro Gly Trp Lys Gly Pro Tyr Cys Thr Glu Pro 190 195 200 IleCys Leu Pro Gly Cys Asp Glu Gln His Gly Phe Cys Asp Lys Pro 205 210 215Gly Glu Cys Lys Cys Arg Val Gly Trp Gln Gly Arg Tyr Cys Asp Glu 220 225230 235 Cys Ile Arg Tyr Pro Gly Cys Leu His Gly Thr Cys Gln Gln Pro Trp240 245 250 Gln Cys Asn Cys Gln Glu Gly Trp Gly Gly Leu Phe Cys Asn GlnAsp 255 260 265 Leu Asn Tyr Cys Thr His His Lys Pro Cys Lys Asn Gly AlaThr Cys 270 275 280 Thr Asn Thr Gly Gln Gly Ser Tyr Thr Cys Ser Cys ArgPro Gly Tyr 285 290 295 Thr Gly Ala Thr Cys Glu Leu Gly Ile Asp Glu CysAsp Pro Ser Pro 300 305 310 315 Cys Lys Asn Gly Gly Ser Cys Thr Asp LeuGlu Asn Ser Tyr Ser Cys 320 325 330 Thr Cys Pro Pro Gly Phe Tyr Gly LysIle Cys Glu Leu Ser Ala Met 335 340 345 Thr Cys Ala Asp Gly Pro Cys PheAsn Gly Gly Arg Cys Ser Asp Ser 350 355 360 Pro Asp Gly Gly Tyr Ser CysArg Cys Pro Val Gly Tyr Ser Gly Phe 365 370 375 Asn Cys Glu Lys Lys IleAsp Tyr Cys Ser Ser Ser Pro Cys Ser Asn 380 385 390 395 Gly Ala Lys CysVal Asp Leu Gly Asp Ala Tyr Leu Cys Arg Cys Gln 400 405 410 Ala Gly PheSer Gly Arg His Cys Asp Asp Asn Val Asp Asp Cys Ala 415 420 425 Ser SerPro Cys Ala Asn Gly Gly Thr Cys Arg Asp Gly Val Asn Asp 430 435 440 PheSer Cys Thr Cys Pro Pro Gly Tyr Thr Gly Arg Asn Cys Ser Ala 445 450 455Pro Val Ser Arg Cys Glu His Ala Pro Cys His Asn Gly Ala Thr Cys 460 465470 475 His Glu Arg Gly His Arg Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly480 485 490 Gly Pro Asn Cys Gln Phe Leu Leu Pro Glu Leu Pro Pro Gly ProAla 495 500 505 Val Val Asp Leu Thr Glu Lys Leu Glu Gly Gln Gly Gly ProPhe Pro 510 515 520 Trp Val Ala Val Cys Ala Gly Val Ile Leu Val Leu MetLeu Leu Leu 525 530 535 Gly Cys Ala Ala Val Val Val Cys Val Arg Leu ArgLeu Gln Lys His 540 545 550 555 Arg Pro Pro Ala Asp Pro Cys Arg Gly GluThr Glu Thr Met Asn Asn 560 565 570 Leu Ala Asn Cys Gln Arg Glu Lys AspIle Ser Val Ser Ile Ile Gly 575 580 585 Ala Thr Gln Ile Lys Asn Thr AsnLys Lys Ala Asp Phe His Gly Asp 590 595 600 His Ser Ala Asp Lys Asn GlyPhe Lys Ala Arg Tyr Pro Ala Val Asp 605 610 615 Tyr Asn Leu Val Gln AspLeu Lys Gly Asp Asp Thr Ala Val Arg Asp 620 625 630 635 Ala His Ser LysArg Asp Thr Lys Cys Gln Pro Gln Gly Ser Ser Gly 640 645 650 Glu Glu LysGly Thr Pro Thr Thr Leu Arg Gly Gly Glu Ala Ser Glu 655 660 665 Arg LysArg Pro Asp Ser Gly Cys Ser Thr Ser Lys Asp Thr Lys Tyr 670 675 680 GlnSer Val Tyr Val Ile Ser Glu Glu Lys Asp Glu Cys Val Ile Ala 685 690 695Thr Glu Val 700

What is claimed:
 1. A DNA coding at least amino acid sequence of the sequence listing, SEQ ID NO:
 1. 2. The DNA according to claim 1 coding at least amino acid sequence of the sequence listing, SEQ ID NO:
 2. 3. The DNA according to claim 1 coding at least amino acid sequence of the sequence listing, SEQ ID NO:
 3. 4. The DNA according to claim 1 having base sequence from No. 355 to 927 of the a sequence listing, SEQ ID NO:
 4. 5. The DNA according to claim 2 having base sequence from No. 355 to 1854 of the sequence listing, SEQ ID NO:
 4. 6. The DNA according to claim 3 having base sequence from No. 355 to 2331 of the sequence listing, SEQ ID NO:
 4. 7. A recombinant DNA comprising a DNA selected from the group consisting of the DNAs of claims 1, having ligated thereto a vector DNA which can express said DNA in the host cell.
 8. A cell transformed by the recombinant DNA of claim
 7. 9. A method for culturing human cells with the cells of claim
 8. 10. A process for production of a polypeptide having at least amino acid sequence of the sequence listing, SEQ ID NO: 1, comprising culturing the cells of claim 8 and isolating the compound produced in the cultured mass.
 11. An antibody specifically recognizing a polypeptide having amino acid sequence of the sequence listing, SEQ ID NO:
 3. 